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

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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

attractive because of the properties of the mesoporous materials.
Cisplatin and its analogs are among the most used antitumor MCM-41-type particles have an ordered arrangement of uniform
drugs for clinical chemotherapy [1,2]. However, small molecule has two-dimensional (2D) hexagonal p6m mesopores [15]. They have
side effects such as kidney toxicity, nausea, hearing impairment, been proved to be nontoxic, pore size adjustable, physicochemi-
and irreversible peripheral nerve damage which caused much pain cally stable, easy modified and functionalized [16,17]. Especially,
to patients [3,4]. To reduce the toxicities of platinum drugs, drug their high surface areas and straight narrow channels facilitate
delivery systems are developed with a capacity of passive tumor adsorption of drugs into their structures [18]. There are some
targeting which enables platinum drugs being directed to solid reports on the loading cisplatin onto the MSN to enhance the
tumors through the enhanced permeability and retention effect cytotoxic effect [19,20]. For instance, Gu et al. [21] prepared MSN
(EPR) [5,6]. The drug carriers could refrain drug from undesired nanocarriers with high-density of carboxyl groups to combine
release before reaching tumor, and release drug in a controlled and with platinum atoms in cisplatin. The complexes greatly improved
smart manner. Several type of nanocarriers have been investigated growth inhibition effect against MCF-7 and HeLa cells. However,
such as polymer micelles [7,8], polymer capsules [9] and inorganic once again cisplatin could cause serious side effects and resistance
particles [10–14]. from cancer cells. It is still a big challenge to develop a safer and
Over the past decade, especially in recent years, mesoporous more effective drug delivery system for cancer therapy.
silica nanoparticles (MSN) have attracted much attention because Oxaliplatin, oxalate (trans-1,2-diaminocyclohexane) plat-
of their excellent properties. Especially, the examples derived 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
∗ Corresponding author. Tel.: +86 431 85262769; fax: +86 431 85262769. [22]. The study on structure–activity relationship of oxaliplatin has
E-mail address: ybhuang@ciac.jl.cn (Y. Huang). 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 2. Experimental
in the oxaliplatin molecule [23]. Numerous nanocarriers have
been employed to form complexes with active anticancer species 2.1. Materials
(DACH-Pt) of oxaliplatin [10,12,24–26]. For instance, Xiao et al.
[7] synthesized a biodegradable and amphiphilic copolymer that Tetraethyl orthosilicate (TEOS, Aladdin), n-cetyltr-
contains pendant 1,2-bidentate carboxyl groups as chelator for imethylammonium bromide (CTAB, YiLi Fine Chemicals Co.
platinum atom. The polymer platinum complex can self-assemble Ltd., Beijing), 3-mercaptopropyltrimethoxysilane (MPTMS,
into micelles and act as pro-drug of oxaliplatin. However, to our Aladdin), (3-aminopropyl) trimethoxysilane (APTES, Sigma),
best knowledge, there is no report on loading active species of maleic anhydride (Aladdin) were used as received. ((1R,2R)-1,2-
oxaliplatin using MSN. Diaminecyclohexano) platinum(II) dinitrate (DACH-Pt-(NO3 )2
Inspired by the above-mentioned work, we for the first time for short) was synthesized as reported [30]. Tetrahydrofuran
designed and synthesized an MSN–oxaliplatin conjugates (MSN- (THF) and toluene were purified by distillation from sodium
Pt) by reaction of active anticancer species (DACH-Pt) of oxaliplatin with benzophenone. 2-(4-Amidinophenyl)-6-indolecarbamidine
with 1,2-bidentate carboxyl-modified MSN. To make a complex dihydrochloride (DAPI), fluorescein isothiocyanate (FITC) and
with platinum atom, carboxylic groups were essential for MSN. Sev- 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide
eral methods were reported to prepare carboxylic-functionalized (MTT) were purchased from Sigma–Aldrich Co. Lyso-tracker Red,
mesoporous silica [27]. One attractive protocol was developed Enhanced BCA Protein Assay Kit and Genomic DNA Mini Prepara-
by Yan et al. [28]. They introduced a high density of carboxyl tion Kit with Spin Column were purchased from Beyotime Institute
groups to MSN by a co-condensation method using tetraethyl of Biotechnology. Other reagents were commercially available and
orthosilicate (TEOS) and functional silane precursor which bears used as received.
upon itself a reactive anhydride group. The precursor was synthe-
sized via an efficient thiol-ene addition reaction which is featured 2.2. Characterizations
by high yields, high regioselectivity and lack of sensitivity to
water and oxygen [29]. However, the functional silane precur- Transmission electron microscopy (TEM) studies were per-
sor needed to be purified by chromatography on silica gel using formed on a JEM-1011 electron microscope operating at an acceler-
dry dichloromethane as eluent before further use, which was an ation voltage of 100 kV. N2 adsorption–desorption isotherms were
extra cost of time and money. Besides, the carboxyl groups func- recorded on a Micromeritics ASAP 2020M automated sorption ana-
tional MSN were not used in drug delivery systems. Herein, we lyzer. The samples were degassed at 150 ◦ C for 5 h. The specific
introduced a more modified, simpler and more convenient route surface areas were calculated from the adsorption data in the low
which was applied to prepare MSN modified with 1,2-bidentate pressure range using the BET model and pore size was determined
carboxyl groups as platinum nanocarriers (Fig. 1A). First, parent following BJH method. Fourier-transform infrared (FT-IR) spectra
particles MSN with thiol groups on the walls (MSN-SH) were syn- were recorded with KBr pellets on a Bio-Rad Win-IR instrument
thesized by one-pot method. Subsequently, the thiol groups reacted in the range from 4000 to 400. The solid-state 13 C and 29 Si CP
with maleic anhydride via efficient thiol-ene addition reaction to MAS NMR spectra were recorded on a Bruker AVANCE III 400 WB
introduce a high density of 1,2-bidentate carboxyl groups. All puri- spectrometer at 100.62 MHz and 79.50 MHz, respectively. X-ray
fied steps could be easily realized by centrifugation. Thereafter, the measurements (XRD) were performed on a Bruker D8 FOCUS Pow-
pendant 1,2-bidentate carboxyl group on prepared nanocarriers der X-ray Diffractometer using Cu K␣ radiation. Inductively coupled
could act as a chelator for the active anticancer species (DACH-Pt) plasma mass spectrometry (ICP-MS, Xseries II, Thermoscientific,
of oxaliplatin to form the oxaliplatin loaded nanoparticles MSN-Pt USA) was used for quantitative determination of platinum.
(Fig. 1B). Moreover, MSN-Pt can be endocytosed by the cancer cells.
After that, they can release clinic relevant active anticancer species 2.3. Synthesis of MSN-COOH
of oxaliplatin which would undergo further binding with cellular
DNA and the formation of 1, 2-(GpG) crosslinks to inhibit cancer The parent particles MSN-SH modified with thiol groups were
cell replication. 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
Fig. 1. (A) Modification of 1,2-bidentate carboxyl groups onto the parent nanoparti-
were collected by centrifugation and washed with water to remove
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 the redundant Na2 CO3 . Thereafter, the particles were mixed with
mediated hydrolysis to inhibit the replication of DNA. 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 77

the solution was stirred vigorously for 48 h at 25 ◦ C in a dark and for 30 min at room temperature. After staining with Lyso-tracker
nitrogen atmosphere. The resulted particles were separated by Red for endosomes and lysosomes and DAPI for the nucleus, the
centrifugation and washed with deionized water to remove the slides were mounted and observed with an Olympus FV1000 con-
unreacted platinum complex. The obtained conjugates were named focal laser scanning microscope (CLSM) imaging system (Japan).
as MSN-Pt.
2.10. Bioactive platinum content in cells
2.5. Synthesis of FITC labeled MSN-Pt (MSN Pt/FITC)
The content of bioactive platinum which was binding with pro-
The MSN-Pt were labeled with FITC according to a previously
tein and DNA in HepG-2 cells was examined by direct platinum
reported method [32]. Briefly, FITC (5 mg) and APTES (10 ␮L) were
measurements using ICP-MS. HepG-2 cells were seeded in 6-well
dissolved in 5 mL of ethanol and the mixture was stirred under
plates. These cells were then treated with oxaliplatin or MSN-Pt
dark for 2 h to obtain FITC-APTES stock solution. Next, 30 mg of
with the platinum concentration of 10 ␮M and subsequently incu-
MSN-Pt was added to the solution. After the reaction proceeded for
bated at 37 ◦ C for determined time. After washing with PBS three
another 24 h, the particles was collected by centrifugation, dialyzed
times, cells were lysed by cell lysis buffer. The platinum contents
against deionized water for 48 h and then freeze-dried to obtain
in each cell lysis sample were determined by ICP-MS. The corre-
MSN-Pt/FITC.
sponding protein content in cells was determined by enhanced
bicinchoninic acid (BCA) assay. The total platinum content was
2.6. In vitro drug release
expressed as ng Pt/mg Protein. In order to evaluate the platinum
contents adducted with DNA, DNA was isolated by using DNA
To evaluate the drug release properties, MSN-Pt (4 mg) was dis-
purification kit after cells were lysed, and DNA was digested with
persed in 5 mL of phosphate buffer (pH 7.4) or acetate buffer (pH
DNase I before analysis. Total Pt–DNA adducts were expressed as
5.0) solutions. The suspensions were then transferred into a pre-
ng Pt/␮g DNA.
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 3. Results and discussion
was withdrawn from the release medium, and the same volume of
fresh buffer was added to the incubation medium. The taken out 3.1. Synthesis and characterization of MSN-COOH
dialysis medium was measured by ICP-MS to calculate the released
amount of platinum. To prepare MSN modified with 1,2-bidentate carboxyl groups as
platinum nanocarriers, we introduced a convenient route as illus-
2.7. Cell lines trated in Fig. 1A. Firstly, parent particles MSN with thiol groups
on the walls (MSN-SH) were synthesized by one-pot method. Sub-
HepG-2 cancer cells (a Human liver cancer cell line) was kindly sequently, we introduce a high density of 1,2-bidentate carboxyl
supplied by the Medical Department of Jilin University in China groups via efficient thiol-ene addition reaction according to the
and first cultured in Dulbecco’s modified Eagle’s medium (DMEM, literature [28]. The advantage of our protocol lies in that it is unnec-
GIBCO), supplemented with 10% heat-inactivated fetal bovine essary to synthesize the functional silane precursor during which
serum (FBS, GIBCO), 100 U/mL penicillin and 100 ␮g/mL strepto- the purifying process is a waste of time. In our method, in fact, syn-
mycin at 37 ◦ C in a humidified atmosphere containing 5% CO2 . thesis of MSN-COOH is efficiency and all purified steps could be
easily realized by centrifugation.
2.8. Cytotoxicity of the MSN-COOH and MSN-Pt The parent MSN-SH was prepared by a one-pot, sol–gel
method as reported [31]. The morphology and pore structures
The in vitro cytotoxicity of the empty particles MSN-COOH and of the parent MSN-SH were characterized by TEM and N2
the conjugates MSN-Pt were evaluated by MTT assay against HepG- adsorption–desorption. It was clearly observed that MSN-SH were
2 cells. Briefly, cells harvested in a logarithmic growth phase were uniform spherical particles with a mean diameter of approximately
seeded in 96-well plates at a density of 105 cells/well and incu- 100 nm (Fig. 2A), and the mesopore channels showed well-ordered
bated in 100 ␮L of DMEM for 24 h. The medium was then replaced 2D hexagonal patterns. The BET isotherm exhibited the characteris-
by empty particles MSN-COOH, oxaliplatin and MSN-Pt at a series tic type of IV N2 adsorption/desorption patterns (Fig. 2B), indicating
of final equivalent platinum concentration. The incubation was that the particles possessed uniform mesoporous channels and
continued for 48 h. Then, 20 ␮L of MTT solution in PBS with the narrow pore size distribution [33]. The surface area and cumula-
concentration of 5 mg/mL was added and the plates were incu- tive pore volume were calculated to be 965 m2 /g and 1.401 cm3 /g,
bated for another 4 h at 37 ◦ C, After that, the medium containing respectively, and the average pore size was about 2.75 nm accord-
MTT was removed and 150 ␮L of DMSO was added to each well to ing to the BJH pore size distribution. These data indicated that the
dissolve the MTT formazan crystals. Finally, the plates were shaken MSN-SH of a mesoporous quality was successfully prepared.
for 10 min, and the absorbance of formazan product was measured In order to introduce the 1,2-bidentate carboxyl groups, thiol
at 492 nm by a microplate reader. groups on the silica particle walls were used to react with maleic
anhydride easily and efficiently via thiol-ene addition reaction.
2.9. Internalization and distribution in cells 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
The internalization and distribution in cells of MSN-Pt/FITC was MSN-COOH showed additional resonance signals at about 36,
determined by CLSM toward HepG-2 cells. Cells were seeded in 6- 69 and 176 ppm, which were assigned to characteristic carbon
well culture plates (a sterile coverslip was put in each well) at a peaks on 1,2-bidentate carboxyl groups. Meanwhile, the 29 Si solid-
density of 5 × 104 cells/well and allowed to adhere for 24 h. Then state NMR spectrum of MSN-COOH presented the bulk silicon
cells were incubated with free MSN-Pt/FITC at a final Pt concen- peaks (Q region) around −105 ppm and two signals at −58 and
tration of 100 ␮g/mL for 0.5, or 4 h at 37 ◦ C. After the preset time −67 ppm corresponding to the functionalized silica resonances (T
intervals, the culture medium was removed and cells were washed region), indicating that organic groups were covalently attached
for three times with ice-cold PBS and fixed with 4% formaldehyde 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

relationship relying on that 1,2-diaminocyclohexane works as a
stable ligand of Pt atom while the oxalic acid is a leaving ligand in
Fig. 3. 13 C CP-MAS solid-state NMR spectrum of MSN-SH (a) and MSN-COOH (b); the oxaliplatin molecule. Also, the 1,2-diaminocyclohexane plat-
29
Si CP-MAS solid-state NMR spectrum of MSN-COOH (c). 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 79

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- diffusion, the MSN-Pt conjugates might be taken up through a more
latin molecular structure conjugating with silica particles. Most efficient endocytosis route and then hydrolyzed in late endosomes
importantly, this structure could make sure to release the active or lysosomes. This different route might result in a higher con-
anticancer species (DACH-Pt) from the conjugates in a denser acidic centration of active DACH-Pt species in cells, which have a higher
condition inside cancer cells. cytotoxicity capacity than oxaliplatin. This could be further proved
After conjugated with platinum drugs, IR spectrum of MSN by cellular internalization experiments in the next section.
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 3.4. Internalization and distribution in cells
group was completely converted to carboxylate ions (Fig. 4A) [28].
In addition, the XPS spectra of MSN-Pt as well as the carrier MSN- Previous reports suggested that MSN were taken up into cells
COOH were conducted. As shown in Fig. 5A, the spectra of MSN-Pt via endocytosis and mainly located in the acidic compartments
exhibited two strong Pt4f signals in comparison with MSN-COOH. such as endosomal and lysosomal vesicles which should facili-
This suggested that the conjugates were successfully prepared. The tate the release of the anticancer drug [39,40]. To correlate the
platinum content of MSN-Pt was determined to be 7.9 wt% by ICP- improved cytotoxicity of the MSN-Pt that was mentioned above,
MS, corresponding to a high equivalent loading of oxaliplatin of a co-localization study was carried out to determine whether the
16.1%. conjugates accumulated within the lysosomes and endosomes after
The release profile of platinum drugs from the MSN-Pt in acetate endocytosis. After cells were incubated with MSN-Pt/FITC for 1 h
buffer solution (pH 5.0) and PBS (pH 7.4) were evaluated by dialysis or 4 h, the endosomes and lysosomes were labeled with red flores-
method. The release amount of platinum was determined by ICP- cent Lyso-tracker Red. As shown in Fig. 7, the CLSM images clearly
MS, and the weight ratio of cumulative released platinum to the revealed that after 1 h of incubation, the green FITC fluorescence
total platinum payload in the MSN-Pt conjugates was calculated. appeared within HepG-2 cells. With the incubation time prolonged
A sustained and pH-sensitive release manner was demonstrated to 4 h, both the green and red fluorescence became stronger and
(Fig. 5B). Nearly 70% of the loaded platinum was released after 24 h they were finely co-localized. It implied that the conjugates could
at pH 5.0, while at pH 7.4 the platinum release was only 20%. The be taken up by cells through endocytosis efficiently and concen-
faster release rate under acidic conditions was the result of the pro- trated within endosomes and lysosomes of HepG-2 cells. Under
tonation of the carboxyl groups, which lead to the dissociation of the acidic environment of the lysosomes, the active DACH-Pt could
active anticancer species DACH-Pt. This is beneficial to intracellu- subsequently be released quickly, which might be the reason for
lar release of anticancer drugs due to the acidic environment in the improved cytotoxicity of the MSN-Pt conjugates.
endosomes and lysosomes (pH 5.0–5.5).
3.5. Bioactive platinum content in cells
3.3. Cytotoxicity assay

To confirm the improved cytotoxicity of the MSN-Pt conju-

The in vitro cytotoxicities of free oxaliplatin and MSN-Pt were
gates more directly, the amount of platinum which was uptaken
examined by MTT assay. HepG-2 cells were selected and cultured
by HepG-2 cells and adducted with DNA, was assessed by ICP-MS.
with oxaliplatin and MSN-Pt in a series of doses (6.25, 12.5, 25,
The platinum content is expressed as “ng of platinum per mg of
50, 100 and 200 ␮M of Pt) for 48 h. The blank particle MSN-COOH
proteins” and “pg of platinum per ␮g of DNA”. The results were
was similarly examined to evaluate its safety. The results are pre-
shown in Table 1. When cells were incubated with oxaliplatin and
sented in Fig. 6. First of all, the cell viability after 48 h incubation
MSN-Pt at an equivalent initial platinum concentration of 0.25 ␮M
with the blank particles was above 90% even at the highest concen-
for 1 h, the platinum contents in cells were 0.14 and 42.52 ng of
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 Table 1
dose-dependent cytotoxicity toward the HepG-2 cells (Fig. 6B). It The contents of bioactive platinum drugs in HepG-2 cells.
is worth noting that the IC50 (the half maximal inhibitory concen- Sample Cell uptake [ng Pt adduct [pg
tration of drug) of MSN-Pt and free oxaliplatin were 25.95 ␮M and Pt/mg Protein] Pt/␮g DNA]
39.29 ␮M, respectively, meaning that MSN-Pt exhibited a some- 1h 4h 24 h
what higher cytotoxicity. The improved cytotoxicity is supposed to
Oxaliplatin 0.14 0.69 2.72
be caused by a totally different mechanism of internalization. In
MSN-Pt 42.52 80.11 9.53
contrast to free oxaliplatin which is known to enter cells by passive
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 drug carriers MSN-COOH bearing 1,2-bidentate carboxyl groups
to 4 h, the platinum contents in cells increased to 0.69 and 80.11 ng were synthesized via a simple and convenient protocol, and subse-
of Pt/mg Protein, respectively. These data directly indicated that the quent conjugation with platinum drugs were successful to give the
platinum delivery through MSN-Pt conjugates into cells has much direct binding of oxaliplatin molecules with MSN. The conjugates
higher efficiency than that of oxaliplatin for HepG-2 cells. were taken up fast by HepG-2 cells and had concentrated within
It is well-known that platinum(II) drugs exert their cytotoxicity endosomes and lysosomes. They displayed an improved cytotox-
by binding DNA and cause apoptosis of cancer cells. The amount of icity against HepG-2 cells than that of free oxaliplatin. The higher
platinum to form Pt-DNA adducts is the most direct measurement level of platinum content both in cells and DNA might be the reason
of drug bioactivity. We incubated HepG-2 cells with free oxalip- of their improved therapeutically effective. These results support
latin and MSN-Pt conjugates for 24 h, followed by collecting and that the platinum loaded MSN conjugates should have great poten-
purifying the total DNA of the treated cells. For free oxaliplatin tial applications in cancer therapy with reduced side effects and
and MSN-Pt conjugates groups, the platinum contents in DNA were enhanced therapeutical efficacy.
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 Acknowledgments
effective. More important, the enhanced accumulation of platinum
content in cells and DNA samples has the obvious potential in solv- The authors would like to thank the financial support from
ing platinum drug resistance. National Natural Science Foundation of China (Nos. 51321062,
51273194 and 21174143), the Ministry of Science and Technol-
4. Conclusions ogy of China (973 Project, No. 2009CB930102; 863 Project, No.
2012AA021900), “100 Talents Program” of the Chinese Academy
In this paper, we prepared MSN-Pt conjugates based on oxalipla- of Sciences (No. KGCX2-YW-802), and Jilin Provincial Science and
tin drugs with mesoporous silica nanoparticles as drug carriers. The Technology Department (No. 20100588).
 H. He et al. / Colloids and Surfaces B: Biointerfaces 117 (2014) 75–81 81

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