Biomaterials 76 (2016) 87e101

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

Rational design of multifunctional magnetic mesoporous silica

nanoparticle for tumor-targeted magnetic resonance imaging and
precise therapy
Wei-Hai Chen a, 1, Guo-Feng Luo a, 1, Qi Lei a, Feng-Yi Cao a, Jin-Xuan Fan a, Wen-Xiu Qiu a,
Hui-Zhen Jia a, Sheng Hong a, Fang Fang b, Xuan Zeng a, Ren-Xi Zhuo a,
Xian-Zheng Zhang a, *
a
Key Laboratory of Biomedical Polymers of Ministry of Education & Department of Chemistry, Wuhan University, Wuhan 430072, PR China
b
State Key Laboratory of Magnetic Resonance and Atomic and Molecular Physics, National Center of Magnetic Resonance in Wuhan, Wuhan Institute of
Physics and Mathematics, Chinese Academy of Sciences, Wuhan 430071, China

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

Article history: In this paper, a multifunctional theranostic magnetic mesoporous silica nanoparticle (MMSN) with
Received 15 October 2015 magnetic core was developed for magnetic-enhanced tumor-targeted MR imaging and precise therapy.
Accepted 21 October 2015 The gatekeeper b-cyclodextrin (b-CD) was immobilized on the surface of mesoporous silica shell via
Available online 22 October 2015
platinum(IV) prodrug linking for reduction-triggered intracellular drug release. Then Arg-Gly-Asp (RGD)
peptide ligand was further introduced onto the gatekeeper b-CD via hosteguest interaction for cancer
Keywords:
targeting purpose. After active-targeting endocytosis by cancer cells, platinum(IV) prodrug in MMSNs
Mesoporous silica nanoparticle
would be restored to active platinum(II) drug in response to the innative reducing microenvironment in
Tumor-targeted
Magnetic resonance imaging
cancer cells, resulting in the detachment of b-CD gatekeeper and thus simultaneously triggering the in
Theranostic situ release of anticancer drug doxorubicin (DOX) entrapped in the MMSNs to kill cancer cells. It was
Precise therapy found that with the aid of an external magnetic field, drug loaded MMSNs showed high contrast in MR
imaging in vivo and exhibited magnetically enhanced accumulation in the cancer site, leading to sig-
nificant inhibition of cancer growth with minimal side effects. This multifunctional MMSN will find great
potential as a theranostic nanoplatform for cancer treatment.
© 2015 Elsevier Ltd. All rights reserved.

1. Introduction
In conventional cancer therapy, chemotherapeutic drugs
without the capability to discriminate between cancerous and
normal cells always result in undesirable side effects and high
systemic toxicity. To achieve satisfactory therapeutic index of drugs,
nanoplatform based drug carriers with passive tumor targeting
ability via the enhanced permeability and retention (EPR) effect
have attracted increasing attention in cancer therapy [1e4].
Nevertheless, the EPR efficacy is always limited by the special tu-
mor microenvironment, such as dense stroma, mutational extra-
cellular matrix, increased interstitial fluid pressure, and
pathophysiological heterogeneity of tumor [5e7]. In addition,
* Corresponding author.
E-mail address: xz-zhang@whu.edu.cn (X.-Z. Zhang).
1
These authors contributed equally to this work.
although the EPR effect can increase the accumulation of nano-
medicines in tumor tissues, the poor cellular internalization and
insufficient intracellular drug release also hamper the utilization of
anticancer drugs, leading to limited therapeutic effect [8,9]. To
overcome these barriers, the combination of multiple functional-
ities into the nanocarrier is highly desired, which relies on the
rational design of sophisticated drug delivery nanosystem [10,11].
Particularly, theranostic nanoplatform with diagnostic and thera-
peutic capabilities offers great opportunities to revolutionize can-
cer therapy. The imaging-guided therapy is beneficial to accurately
destroy cancer and realize precise therapy [12].
Recently, mesoporous silica nanoparticles (MSNs) have
garnered prominent research interest as drug nanocarriers due to
their intrinsically favorable biocompatibility, tunable pore sizes,
large loading capacity, and convenient surface functionalization
[13e16]. For instance, intelligent molecular machines which
respond to endogenous stimuli, including redox, pH, temperature

http://dx.doi.org/10.1016/j.biomaterials.2015.10.053
0142-9612/© 2015 Elsevier Ltd. All rights reserved.
88 W.-H. Chen et al. / Biomaterials 76 (2016) 87e101

and enzymes, have been employed on MSNs to control the drug
release extensively [17e22]. Despite these encouraging achieve-
ments, mechanized MSNs have suffered disadvantages since the
use of stimuli are complicated to apply in vivo or not available
innately in the targeted tumor cells as well as lack of diagnostic
specificity [23]. To improve the applicability and further function-
alize the MSNs-based materials, integration of MSNs with inorganic
nanocrystals to form uniform multifunctional nanocomposites has
emerged as one of the most popular research directions recently.
For instance, quantum dots, gold nanoparticles, silver nanoparticles
and magnetic nanoparticles, have been successfully incorporated
within MSNs [24e27]. Of particular significance are magnetic
mesoporous silica nanoparticles [28e33]. The cooperation of
magnetic nanoparticles is beneficial for magnetically targeted drug
delivery as well as an excellent contrast agent for magnetic reso-
nance imaging (MRI), which provides great opportunity for the
construction of nanoscaled theranostic platform for cancer therapy.
Moreover, as a physical force, the magnetic stimulus is independent
of the complicated tumor physiological microenvironment and the
magnetic targeting mediated EPR effect can increase the accumu-
lation of nanoparticles in the targeted tumor site [34e38], which is
rewarding to efficiently and specifically deliver the therapeutic
agents to the tumor region.
In this study, a multifunctional theranostic platform based on
magnetic mesoporous silica nanoparticles (MMSNs) was inge-
niously designed for tumor-targeted magnetic resonance imaging
and precise therapy. As illustrated in Scheme 1, MMSNs with the
superparamagnetic Fe3O4 core and mesoporous silica shell were
employed as MRI contrast agent and anticancer drug nano-
container simultaneously. Doxorubicin (DOX) as a model anticancer
drug was loaded in the porous structure of the MSN shell.
Multifunctional molecular machine on MMSNs was constituted and
constructed by platinum(IV) prodrug, b-cyclodextrin, and cancer-
targeted peptide adamantane-PEG8-glycine-arginine-glycine-
aspartic-serine (AD-PEG8-GRGDS) progressively. The b-CD was
used as the gatekeeper and immobilized onto mesoporous silica
shell via the linker of platinum(IV) prodrug, which was redox-
sensitive and could be intracellularly activated by reduction to
the toxic platinum(II) drug [39,40]. For the first time, platinum(IV)
prodrug was exploited as the redox-sensitive linker to trigger the
drug release from mesoporous silica as well as chemotherapeutic to
assist the ablation of cancer cells. Finally, the cancer active targeting
peptide AD-PEG8-GRGDS was assembled on MMSNs as a targeting
ligand toward cancer cells via hosteguest interaction between AD
and b-CD.
As illustrated in Scheme 1B, after intravenous injection, the
multifunctional MMSNs were expected to accumulate at cancer
sites as a combined function of the magnetic targeting enhanced
EPR effect and the active targeting effect. The magnetic property of
MMSNs would provide great contrasts for magnetic resonance
imaging to localize the cancer site and guide the therapy. On the
other hand, since the RGD motif could targetedly bind avb3 integrin
overexpressed by cancer cells [41,42], multifunctional MMSNs
would preferentially internalize in cancer cells. After uptaken by
cancer cells, the platinum(IV) prodrug conjugated on the surface of
MMSN would be reduced by intracellular reductive microenvi-
ronment and converted to active platinum(II) drug to kill cancer
cells. Importantly, the loaded DOX would be released in situ from
mesoporous silica shell since the b-CD gatekeeper was removed in
the progress of reducing platinum(IV) to platinum(II), producing
cytotoxicity and inducing the death of cancer cells.

Scheme 1. Schematic illustration of the design and proposed mechanism of the multifunctional MMSNs for tumor-targeted MRI and precise therapy. (A) The functionalization
protocol of the MMSN. (B) The precise treatment strategy by using multifunctional MMSNs. Specifically, the magnetic enhanced EPR effect and the active targeting effect facilitated
the enriching of MMSNs in tumor sites; then MMSNs were selectively uptaken by cancer cells via the receptor-mediated endocytosis; afterward the platinum(IV) prodrug was
reduced by the cellular innate reductive environment and induced the removal of gatekeeper, thereby triggering the in situ drug release for precise therapy.
 W.-H. Chen et al. / Biomaterials 76 (2016) 87e101
2. Materials and methods
2.1. Materials
N-Fluorenyl-9-methoxycarbonyl (Fmoc) protected L-amino
acids (Fmoc-Arg(Pbf)-OH, Fmoc-Asp(OtBu)-OH, Fmoc-Ser(tBu)-
OH, and Fmoc-Gly-OH, 2-chlorotrityl chloride resin
(100e200 mesh, loading: 0.6 mmol/g, 1%DVB), benzotriazol-1-yl-
oxytripyrrolidinophosphonium hexafluorophosphate (PyBOP), o-
benzotriazole-N,N,N0 ,N0 -tetramethyluroniumhexafluorophosphate
(HBTU), 1-hydroxybenzotriazole (HOBt), triisopropylsilane (TIS),
and piperidine were purchased from GL Biochem. Ltd. (Shanghai,
China) and used as received. Diisopropylethylamine (DIEA) was
acquired from GL Biochem. Ltd. (Shanghai, China) and used after
distillation. Fmoc-PEG8-COOH was provided by Zhoubei Technol-
ogy Co. Ltd. (Hangzhou, China). Trifluoroacetic acid (TFA),
anhydrous ether, ninhydrin, cisplatin (cis-Pt(NH3)2(Cl)2),
hydrogen peroxide (H2O2), succinic anhydride, ferric chloride, so-
dium acetate, 3-aminopropyltriethoxysilane (APTES), cetyl-
trimethylammonium bromide (CTAB), tetraethylorthosilicate
(TEOS), and ammonium nitrate (NH4NO3) were obtained from
Shanghai Chemical Co. (Shanghai, China) and used directly. Ada-
mantane chloride was purchased from Aladdin Reagent Co. Ltd.
(Shanghai, China). N,N-dimethylformamide (DMF), dimethyl sulf-
oxide (DMSO), methanol and dichloromethane (DCM) were pro-
vided by Shanghai Chemical Co. (Shanghai, China) and distilled
prior to use. Mono-6-ethylenediamine-b-CD was prepared ac-
cording to our previous literature procedures [43]. Doxorubicin
hydrochloride was purchased from Zhejiang Hisun Pharmaceutical
Co. (Zhejiang, China). Fetal bovine serum (FBS), penicillin strepto-
mycin, trypsin, 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetra-
zoliumbromide (MTT), molecular probe (Hoechst 33342), and
Dulbecco's phosphate buffered saline (PBS) were purchased from
Invitrogen (USA). All other reagents and solvents were of analytical
grade and used directly.
2.2. Synthesis of the platinum(IV) prodrug
As shown in Fig. S1, to prepare the platinum(IV) prodrug, the
cisplatin was firstly oxidized by excess hydrogen peroxide to yield
Pt(OH)2. In brief, the mixture solution of cisplatin (cis-
Pt(NH3)2(Cl)2, 1.0 g, 3.33 mmol), hydrogen peroxide (H2O2, 30 wt%,
8 mL) and 90 mL DI water were heated to 50  C and stirred for 6 h in
the dark. Then the mixture was cooled to room temperature and
further stirred overnight. The crude product was obtained by
setting at 4  C for 12 h and filtrating. Then crude product was
washed by ice cold water, ethanol and diethyl ether, respectively.
The product (0.85 g) was obtained as a bright yellow powder after
vacuum dried.
Then Pt(OH)2 reacted with succinic anhydride to afford plati-
num(IV) prodrug. In particular, a DMF solution containing Pt(OH)2
(0.5 g, 1.5 mmol) and succinic anhydride (1 g, 10.0 mmol) was
heated to 70  C and stirred gently for 12 h in the dark. Then the
solvent was removed via vacuum distillation and the product was
obtained by recrystallization from acetone at 20  C and isolated
via filtrating, then vacuum dried. The crude product was purified on
a silica gel column using ethyl acetate: methanol ¼ 4:1 to afford the
pure product platinum(IV) prodrug (0.61 g). The calculated mo-
lecular mass of platinum(IV) prodrug was 531.2 and the ESI-MS
result found to be 532.9 (see Fig. S2).
2.3. Synthesis of cancer-targeted peptide adamantane-PEG8-
glycine-arginine-glycine-aspartic-serine (AD-PEG8-GRGDS)
The peptide derivative adamantane-PEG8-glycine-arginine-
89
glycine-aspartic-serine was prepared as our previously reported
method by standard Fmoc solid phase peptide synthesis [44].
Briefly, peptide chains were grown on 2-chlorotriyl chloride resin.
The coupling of first amino acid conducted 4 equiv (relative to the
substitution degree of resin) Fmoc-protected amino acid and
6 equiv of DIEA in a DMF solution for 2 h. Other amino acid cou-
plings were executed with 4 equiv of Fmoc-protected amino acid,
4 equiv of HBTU, and 6 equiv of DIEA in a DMF solution for 4 h.
During the synthesis, Fmoc protected groups were removed by 20%
piperidine/DMF (v/v) for twice and every time for 15 min. At the
end of the synthesis, adamantane chloride was conjugated to the
peptide segments as the similar method. Then the resin was
washed with DMF (four times), methanol (four times) and DCM
(four times), respectively, and then dried under vacuum overnight.
Cleavage of the expected peptide and the removal of side chain
protected groups from the dried resin were performed by sus-
pending the resin in a cleavage cocktail containing TFA (95%), TIS
(2.5%), and H2O (2.5%) for 2 h. The filtration was concentrated to a
viscous solution by rotary evaporation. Then viscous filtration was
precipitated in cold ether to obtain the crude product. After vacuum
drying, the crude product was dissolved in deionized water and
freeze-dried. The molecular weight was determined by ESI-MS
(Finnigan LCQadvantage). The calculated molecular mass of AD-
PEG8-GRGDS was 1075.6 and the ESI-MS result found to be 1075.5
(see Fig. S3).
2.4. Synthesis of the coreeshell type magnetic mesoporous silica
nanoparticles (MMSNs)
The superparamagnetic Fe3O4 core particles were synthesized
through a solvothermal reaction as previously reported [45]. Then
the coreeshell type magnetic mesoporous silica nanoparticles were
further prepared according to the previous reports with slight
modifications [33]. Briefly, Fe3O4 particles were dispersed in chlo-
roform with a concentration of 15.6 mg Fe3O4/mL. Firstly, 1.0 mL
Fe3O4 particles were added into 5 mL CTAB solution (0.55 M) and
the resulted solution was further stirred at 1000 rpm for 20 min.
Then the mixture was heated to 60  C and stirred for another
10 min to evaporate the chloroform. Afterwards, a mixture of 45 mL
deionized water and 0.3 mL NaOH (0.2 M) was added to the above
solution and the resulted solution was heated to 70  C under stir-
ring at 400 rpm. Then 40 mL TEOS was added to the reaction solu-
tion drop by drop and repeated one time after 1 h. After stirring
another 2 h, the reaction was completed and the MMSN was ob-
tained after washing six times with water to remove the unreacted
species.
2.5. Synthesis of MMSN-NH2
MMSN (300.0 mg) was dispersed in anhydrous methanol
(50 mL). Then 3-aminopropyltriethoxysilane (4.5 mL) was added
and the mixture was stirred for 24 h at room temperature. After
centrifugation, the nanoparticles were extensively washed with
methanol and the obtained sample was MMSN-NH2.
2.6. Synthesis of MMSN-NH-Pt-COOH
MMSN-NH2 was conjugated with platinum(IV) prodrug to pre-
pare MMSN-NH-Pt-COOH using a standing coupling procedure.
Briefly, MMSN-NH2 (200.0 mg), platinum(IV) prodrug (300.0 mg,
0.56 mmol), DIEA (0.98 mL, 5.6 mmol), PyBOP (437.1 mg,
0.84 mmol) and HOBt (113.5 mg, 0.84 mmol) were dissolved in the
redistilled DMF (40 mL) and then stirred 24 h at room temperature
in a nitrogen round-bottom flask. The template of CTAB was
removed by refluxing with a methanol solution of NH4NO3 (60 mL,
90 W.-H. Chen et al. / Biomaterials 76 (2016) 87e101
10 mg/mL) through ion exchange for 12 h. Then MMSN-NH-Pt-
COOH nanoparticles were obtained by centrifugation and washed
six times with methanol.
2.7. Synthesis of MMSN-NH-Pt-CD
MMSN-NH-Pt-COOH was reacted with mono-6-
ethylenediamine-b-CD to prepare MMSN-NH-Pt-CD using a
standing coupling procedure. In brief, MMSN-NH-Pt-COOH
(100 mg), mono-6-ethylenediamine-b-CD (300.0, mg), NHS
(172.5 mg) and 191.7 mg EDC (287.5 mg) were dispersed in 25 mL
deionized water and stirred for 48 h in the dark at room temper-
ature. Then MMSN-NH-Pt-CD was obtained after centrifugation
and washed thoroughly with deionized water, and dried under
vacuum.
2.8. Synthesis of MMSN-NH-Pt-CD/AD-RGD
MMSN-NH-Pt-CD/AD-RGD was prepared through hosteguest
interaction between b-CD and AD. MMSN-NH-Pt-CD (100.0 mg)
was dispersed in 25 mL deionized water and then the peptide AD-
PEG8-GRGDS (50.0 mg) was added. The mixture solution was stir-
red at room temperature for 24 h to obtain MMSN-NH-Pt-CD/AD-
RGD nanoparticles. The nanoparticles were washed thoroughly
with deionized water and dried under vacuum.
2.9. Preparation of DOX-loaded MMSNs
MMSN-NH-Pt-COOH (100.0 mg) with 25 mg DOX was
dispersed in 10 mL PBS and stirred at room temperature for 24 h,
then further immobilized the gatekeeper b-CD on the surface of
nanoparticles for drug loading. To react with mono-6-
ethylenediamine-b-CD, the above particles, mono-6-
ethylenediamine-b-CD (300.0, mg), NHS (172.5 mg) and
191.7 mg EDC (287.5 mg) were dispersed in 25 mL deionized water
and stirred for 48 h in the dark at room temperature. After that,
drug loaded nanoparticles were collected by centrifugation and
washed several times with water to remove the unloaded drug
molecules. The supernatant was collected. The DOX loading con-
tent was determined by fluorescence measurement (Loading
content ¼ (initial weight of drug - weight of drug in supernatant)/
(weight of drug loaded nanoparticles)). The DOX loaded MMSN-
NH-Pt-CD/AD-RGD was prepared similar to the procedure
described above.
2.10. Characterizations of the multifunctional MMSNs
The morphology of Fe3O4 particles and MMSNs were observed
by transmission electronic microscopy (TEM, JEOL-2100). Zeta po-
tential instrument (Nano-ZS ZEM3600, Malvern Instruments Co.
Ltd, UK) and Fourier transform infrared spectroscopy (Per-
kineElmer Spectrum One, USA) were employed to monitor the
reaction processes of MMSNs. The surface area was detected by
BrunauereEmmetteTeller (BET) measurements and the pore size
distribution was obtained by Barrete-Joyner-Halenda (BJH)
(ASAP2020, Micromeritics) analyses. All the samples were degassed
at 120  C for 6 h under nitrogen before measurements. The content
of Fe element and Pt element was determined by inductively
coupled plasma atomic emission spectrometry (ICP-AES). The DOX
concentration was determined by RF-530/PC fluorescence spec-
trofluorophotometer analysis (Shimadzu). All the T2-weighted MR
images were measured with a 7.0 T MR spectrometer (Bruker Inc.,
Billerica, MA, USA).
2.11. Redox-sensitive drug release of MMSN-NH-Pt-CD/AD-RGD
To investigate redox-sensitive drug release of MMSN-NH-Pt-CD/
AD-RGD under intracellular reductive environment, ascorbic acid
was chose as the reduction agent, which has been demonstrated to
be a major substance for the reduction of platinum(IV) prodrug
[46,47]. Then 1 mg of DOX loaded MMSN-NH-Pt-CD/AD-RGD
nanoparticles were incubated in three different release media at
37  C: (1) PBS (pH 7.4) without ascorbic acid, (2) PBS (pH 7.4) with
1 mM ascorbic acid, and (3) PBS (pH 7.4) with 10 mM ascorbic acid,
respectively. The drug concentration in the release medium was
detected by RF5301PC spectrofluorophotometer (Shimadzu) at
given time intervals. The emission and excitation slit widths were
set at 5 nm with lex ¼ 480 nm.
2.12. Cell culture
HeLa (human cervical carcinoma) cancer cells and COS7 (African
green monkey kidney fibroblast cells) normal cells were incubated
in DMEM medium with 10% FBS and 1% antibiotics (penicillin-
streptomycin, 10000 U/mL) at 37  C in a humidified atmosphere
containing 5% CO2.
2.13. Characterization of in vitro tumor-targeted drug delivery of
MMSN-NH-Pt-CD/AD-RGD by CLSM
To confirm the active targeting specificity and magnetically
enhanced drug delivery, we incubated DOX loaded MMSN-NH-Pt-
CD/AD-RGD with HeLa and COS7 cells with or without an external
magnetic field. After the cells treated with DOX loaded MMSN-NH-
Pt-CD/AD-RGD (The concentration of DOX was 2 mg/mL) in the cell
culture media for 1 h or 2 h, the culture medias were removed and
the cells were washed with PBS three times to remove the unen-
docytosed particles. Then the nuclei were stained with Hoechst
33342 at 37  C for 15 min and all cells were observed by CLSM
(Nikon C1-si, Japan).
2.14. Analysis of intracellular redox-sensitive drug release by CLSM
and quantitative evaluating cellular uptake of DOX by flow
cytometry
To further quantitatively evaluate cellular uptake of multifunc-
tional MMSNs and examine the intracellular drug delivery, cells
treated with DOX loaded MMSN-NH-Pt-CD or MMSN-NH-Pt-CD/
AD-RGD were measured by CLSM and flow cytometry. HeLa cancer
cells and COS7 normal cells were seeded in a glass bottom dish at a
density of 1  105 cells/well for 24 h, respectively. Then cells were
incubated with DOX loaded MMSN-NH-Pt-CD or MMSN-NH-Pt-CD/
AD-RGD (The concentration of DOX was 2 mg/mL) in the cell culture
media for 4 h. After removed the culture media and washed with
PBS three times, the nuclei were stained with Hoechst 33342 at
37  C for 15 min. Then all cells were observed by CLSM (Nikon C1-si,
Japan). As the control, the cells were treated with free DOX (2 mg/
mL) and observed by CLSM. Quantitative analysis of the intracel-
lular DOX red fluorescence was determined by flow cytometry (BD
FACSAria TM III, USA). To confirm the internalization of MMSN-NH-
Pt-CD/AD-RGD via the avb3 integrin receptor-mediated endocy-
tosis, a competition assay was performed. HeLa cancer cells were
pre-treated with the excess free RGD peptide (50 mM) for 4 h in
advance. Then the culture media were refreshed and incubated
with MMSN-NH-Pt-CD/AD-RGD (The concentration of DOX was
2 mg/mL) and analyzed by microscopy and flow cytometry just as
the above procedure. The cells without treatment were as the blank
control.
 W.-H. Chen et al. / Biomaterials 76 (2016) 87e101
2.15. Characterization of intracellular distribution of MMSN-NH-Pt-
CD/AD-RGD by TEM
In this work, TEM was applied to monitor the distribution of
MMSN-NH-Pt-CD/AD-RGD within HeLa and COS7 cells. HeLa can-
cer cells and COS7 normal cells were incubated with MMSN-NH-Pt-
CD/AD-RGD (The concentration of DOX was 2 mg/mL) for 12 h. Then
the cells were washed, collected by centrifugation and fixed with
1 mL general fixative (containing 2.5% glutaraldehyde in PBS buffer)
at 4  C and overnight. Cell samples were prepared for TEM obser-
vation according to standard procedures and then detected by us-
ing a electronic microscopy (TEM, JEOL-2100).
2.16. Analysis the intracellular uptake amount of Fe by ICP-AES
HeLa cancer cells and COS7 normal cells were incubated with
MMSN-NH-Pt-CD/AD-RGD (The concentration of DOX was 2 mg/
mL) for 2 h, 4 h, 6 h, and 8 h, respectively. After washed with PBS
buffer three times, the cells were counted and collected. Before
measurement, the cell samples were digested with aqua regia
(Extreme caution is required! Aqua regia is highly corrosive and
damaging to skin and eyes!) and diluted with water to 10 mL.
Triplet parallel samples were performed and the average content of
the intracellular Fe element was analyzed using ICP-AES. The cells
without treatment were as the blank control.
2.17. In vitro cell MR imaging
HeLa cancer cells and COS7 normal cells were respectively
seeded in 6-well plates at a density of 5  105 cells/well for 24 h.
Then the cells were incubated with MMSN-NH-Pt-CD/AD-RGD at
different Fe concentrations (0.0, 0.1, 0.2, 0.4, 0.6 mM) for 4 h. After
washed with PBS buffer three times, the cells were counted and
collected. Then the cells (1  106) were suspended in gelatin (2%,
300 mL) in plastic vials homogenously and the T2-weighted MR
images were measured with a 7.0 T MR spectrometer (Bruker Inc.,
Billerica, MA).
2.18. In vitro cytotoxicity test by MTT assay
In vitro ablation test was performed with HeLa cancer cells and
COS7 normal cells by MTT assay. Briefly, HeLa cancer cells and COS7
normal cells were seeded in 96-well plates at a density of 6000
cells/well, and then cells were incubated in 100 mL DMEM con-
taining 10% FBS and 1% antibiotics for 24 h prior to treating with
DOX loaded MMSN-NH-Pt-CD or MMSN-NH-Pt-CD/AD-RGD in the
presence or absence of an external magnetic field. After co-
incubation for 2 h, the culture medium was replaced with 200 mL
of fresh medium and further incubated 46 h. Then 20 mL MTT so-
lutions (5 mg/mL) was added to each well and incubated for 4 h.
After that, the medium was removed and 200 mL DMSO was added.
The absorbance was measured at 570 nm using a microplate reader
(Bio-Rad, Model 550, USA). The relative cell viability was calculated
as: cell viability ¼ (OD570 (samples)/OD570 (control))  100%, where
OD570 (control) was obtained in the absence of MMSN-NH-Pt-CD or
MMSN-NH-Pt-CD/AD-RGD, and OD570 (samples) was obtained in the
presence of MMSN-NH-Pt-CD or MMSN-NH-Pt-CD/AD-RGD. The
cytotoxicity of DOX unloaded MMSN-NH-Pt-CD or MMSN-NH-Pt-
CD/AD-RGD was also evaluated just like the above procedure. Each
value was averaged from four independent experiments.
2.19. Animals and tumor model
All animal experiments were agreed with institutional animal
use and care regulations from Wuhan University. BALB/c nude mice
91
(5-weeks old, Wuhan University Animal Biosafty Level III Lab,
Wuhan) were used for animal experiments. Subcutaneous tumors
were established by injecting HeLa cancer cells (1  107 cells) into
subcutaneous of female mice. To demonstrate the magnetic tar-
geting enhanced MR imaging of tumors, two tumors were inocu-
lated in the left and right back of the hind leg region. For evaluating
the antitumor efficacy of MMSNs, the tumors were inoculated in
right back of the hind leg region.
2.20. In vivo MR imaging under magnetic targeting
When tumors had grown to approximately 200 mm3 in volume,
the tumor-bearing mice were intravenously injected with 200 mL
MMSN-NH-Pt-CD or MMSN-NH-Pt-CD/AD-RGD (The concentration
of DOX was 2 mg/kg and the corresponding particle concentration
was 3.7 mg/mL). Afterwards, the tumor on the right was exposed to
an external magnetic field while the left was not. After injection
30 min and 90 min, T2-weighted MR images were recorded with a
7.0 T MR spectrometer (Bruker Inc., Billerica, MA, USA).
2.21. Biodistribution of nanoparticles in tumor tissues
When tumors had reached approximately 200 mm3 in volume,
the animals were randomized into three groups: MMSN-NH-Pt-CD,
MMSN-NH-Pt-CD/AD-RGD, and MMSN-NH-Pt-CD/AD-RGD with an
external magnetic field. The mice were sacrificed after 24 h tail vein
injection of nanoparticles at a dosage of 2 mg/kg (DOX). Tumor
tissues and main organs (heart, liver, spleen, lung and kidney) for
each group were harvested for analyzing the biodistribution of
different nanoparticles. The concentrations of Si in tumor tissues
and main organs of each group were measured by ICP-AES.
2.22. In vivo therapy and histochemistry analysis
When the tumor volume approximately reached 50 mm3, HeLa
tumor-bearing mice were studied in five groups (n ¼ 6 per group)
and treated with PBS, free DOX (The concentration of DOX was
2 mg/kg), MMSN-NH-Pt-CD (The concentration of DOX was 2 mg/
kg and the corresponding particle concentration was 3.7 mg/mL),
MMSN-NH-Pt-CD/AD-RGD (The concentration of DOX was 2 mg/kg
and the corresponding particle concentration was 3.7 mg/mL) and
MMSN-NH-Pt-CD/AD-RGD (The concentration of DOX was 2 mg/kg
and the corresponding particle concentration was 3.7 mg/mL) with
an external magnetic field, respectively. Mice in five-treated groups
were injected every two day until the completion of experiments.
The weight of mice and tumor growth was monitored every day.
Tumor size was measured by a caliper and tumor volume was
determined using the following formula V ¼ W2  L/2, where W
and L were the shortest and longest diameters of tumors, respec-
tively. On the day of 12, all mice were sacrificed and the tumors
were excised and weighed. Simultaneously, the main organs (heart,
liver, spleen, lung and kidney) of the mice were also harvested and
used for histology analysis.
The tumor tissues were stained by Prussian blue for evaluating
the level of iron in tumors. To analyze the levels of apoptosis in
tumor cells, tumor tissue sections were stained by terminal deox-
ynucleotidyl transferased dUTP nick end labeling according to the
manufacturer's protocol (Roche, Penzberg, Germany). The nuclei
were labeled with 40 ,6-diamidino-2-phenylindole (DAPI) (Invi-
trogen, USA) and then examined by fluorescence microscopy. To
examine the morphology of tumor cells and evaluate the biocom-
patibility of MMSN, the tumor tissues and the main organs (heart,
liver, spleen, lung and kidney) were stained with hematoxylin and
eosin (H&E) for histological examination.
92 W.-H. Chen et al. / Biomaterials 76 (2016) 87e101

2.23. Statistics analysis
The quantitative data collected were expressed as mean ± S.D.
Statistical significance was analyzed by three-sample Student's
text. Statistical significance was inferred at a value of P < 0.05.
3. Results and discussion
3.1. Synthesis and characterization of multifunctional MMSNs
The coreeshell type MMSNs were synthesized according to
literature with slight modifications and the detail procedure was
described in experimental section [33]. As shown in Fig. 1A, the
monodisperse and uniform MMSNs displayed a typical coreeshell
structure. The core of magnetic iron oxide nanoparticles (see
Fig. S4) were coated with mesoporous silica shell, demonstrating
that the successful preparation of MMSNs. The hydrodynamic size
of MMSNs was 103.2 ± 0.5 nm, analyzing by dynamic light scat-
tering (Fig. 1B). N2 adsorptionedesorption isotherms (Fig. 1C) and
BJH pore-size distribution curves (Fig. 1D) manifested that the
MMSNs have well-developed pore diameter of 2.8 nm. The BET
specific surface area and the total pore volume were 593 m2g1 and
0.94 cm3g1, respectively. These results demonstrated the obtained
MMSNs possess the promising potential as the ideal drug nano-
container, which exhibited uniform nanostructure and large ca-
pacity for drug delivery.
Platinum(IV) prodrug was prepared by oxidizing the cisplatin to
produce Pt(OH)2, followed by reacting with succinic anhydride (see
Fig. S1). The cancer targeting peptide AD-PEG8-GRGDS was syn-
thesized via the mature solid phase peptide synthesis technique in
our lab. The product platinum(IV) prodrug and AD-PEG8-GRGDS
peptide were characterized by electrospray ionization-mass spec-
trometry (ESI-MS). The theoretical molecular weight was 531.2 for
platinum(IV) prodrug and 1075.6 for AD-PEG8-GRGDS peptide,
while the observed molecular weights were 532.9 and 1075.5 (see
Fig. S2 and Fig. S3), respectively, confirming the successful
preparation of target products. As shown in Scheme 1A, the
multifunctional machined MMSNs were ultimately prepared after
loading anticancer drug DOX and modifying with b-CD as the
gatekeeper. Briefly, the detailed synthetic procedure included four
steps: (I) MMSNs were first decorated with 3-
aminopropyltrimethoxysilane to obtain MMSN-NH2, (II) MMSN-
NH2 was further modified with the redox-sensitive linker plati-
num(IV) prodrug via the amidation reaction, denoted as MMSN-
NH-Pt-COOH, (III) The gatekeeper b-CD was immobilized onto the
surface of MMSN-NH-Pt-COOH by the reaction between mono-6-
ethylenediamine-b-CD and carboxyl of platinum(IV) prodrug to
obtain MMSN-NH-Pt-CD, and (IV) The cancer targeting peptide AD-
PEG8-GRGDS was functionalized with MMSN-NH-Pt-CD via
hosteguest interaction between AD and b-CD, denoted as MMSN-
NH-Pt-CD/AD-RGD. The successful introduction of multifunctional
machines on MMSNs was monitored and characterized by Fourier
transform-infrared spectroscopy (FT-IR), size measurement and
zeta potential analysis. As shown in Fig. S5, the disappearance of
three strong bands at 2923, 2853, and 1478 cm1 confirmed that
CTAB molecules were removed thoroughly during the refluxing
process in MeOH/NH4NO3 solution, which were the typical CH2
stretching and bending vibrations of CTAB molecules. The appear-
ance absorption peak of 1420 cm1 corresponded to the C-N
stretching vibration in MMSN-NH2 and the absorption peak at
1650 cm1 and 1300 cm1 displayed the typical structure of COOH
in MMSN-NH-Pt-COOH. Moreover, the hydrodynamic diameter of
MMSNs was increased with the stepwise decoration of different
functional groups and the change of zeta potential of MMSNs was
also related to the incorporation of different moieties (Table 1). The
zeta potential of MMNS-NH2 was þ30.8 mV and decreased
to 33.9 mV after linking with platinum(IV) prodrug with a
multitude of carboxyl on the surface of MMSN-NH-Pt-COOH. After
loading DOX and reacting with the gatekeeper b-CD, the zeta po-
tential of MMSN-NH-Pt-CD increased to 9.5 mV and changed to
3.3 mV since the incorporation of AD-PEG8-GRGDS peptide. As
shown in Fig. S6 and Table S1, the BET pore volume and BJH pore

Fig. 1. Characterization of multifunctional theranostic magnetic mesoporous nanoparticles. (A) Transmission electron microscope (TEM) image of MMSNs. (B) Hydrodynamic size
distribution of MMSNs measured by dynamic light scattering (DLS). N2 adsorption/desorption isotherms (C) and BJH pore size distribution (D) of MMSNs. (E) Field-dependent
magnetization loop of MMSN-NH-Pt-CD/AD-RGD at 300 K. The absence of a hystersis loop indicated the superparamagnetic property of MMSN-NH-Pt-CD/AD-RGD. (F) T2-
weighted MR images of MMSN-NH-Pt-CD/AD-RGD in aqueous solution at different Fe concentrations. (G) T2 relaxation rate of MMSN-NH-Pt-CD/AD-RGD as a function of Fe
concentrations. (H) The drug release profile of DOX loaded MMSN-NH-Pt-CD/AD-RGD in PBS buffer with different ascorbic acid concentrations. Error bars were based on at least
triplicate measurements.
 W.-H. Chen et al. / Biomaterials 76 (2016) 87e101 93

Table 1
Monitoring the modification processes of multifunctional theranostic MMSNs by size measurement and zeta potential analysis. The dates were based on triplicate
measurements.

Sample Zeta potential (mV) Hydrodynamic diameter (nm) PDI

MMSNs 15.2 ± 0.4 103.2 ± 0.5 0.06

MMSN-NH2 30.8 ± 0.5 111.2 ± 0.4 0.18
MMSN-NH-Pt-COOH 33.9 ± 0.6 131.1 ± 0.6 0.11
MMSN-NH-Pt-CD 9.5 ± 0.3 148.7 ± 0.5 0.09
MMSN-NH-Pt-CD/AD-RGD 3.3 ± 0.2 163.8 ± 0.5 0.15

diameter decreased gradually during the functionalization process,
which further confirmed the successful modification. Moreover, the
solid state 13C NMR spectra (see Fig. S7) further demonstrated the
functional components were anchored on the surface of MMSNs.
The stability of MMSN-NH-Pt-CD/AD-RGD at normal physiological
conditions was also monitored by dynamic light scattering (DLS)
detection. As shown in Fig. S8, no obvious changes in hydrodynamic
diameter and polydispersity index (PDI) were observed during four
days, indicating that MMSN-NH-Pt-CD/AD-RGD was stable at
normal physiological conditions. These results confirmed the suc-
cessful preparation of multifunctional MMSNs.
It is well known that magnetic iron oxide core of MMSNs has
multiple functionalities, including MRI and magnetic targeting to
direct drugs to localized lesions [48]. In our designed DOX loading
MMSN-NH-Pt-CD/AD-RGD, the weight ratio of Fe was 4.7% (w/w)
and the weight ratio of Pt was 2.4% (w/w), determining by induc-
tively coupled plasma atomic emission spectrometry (ICP-AES).
According to fluorescence measurement, about 5.4% (w/w) DOX
was loaded into the mesoporous silica shell of MMSN-NH-Pt-CD/
AD-RGD. Field-dependent magnetism loop of drug loaded MMSN-
NH-Pt-CD/AD-RGD at 300 K showed no hysteresis (Fig. 1E), indi-
cating that they have the superparamagnetic property. Further-
more, T2-weighted MRI of MMSN-NH-Pt-CD/AD-RGD on a 7.0 T MR
spectrometer was investigated and utilized to evaluate their
contrast enhancement effect. As shown in Fig. 1F, the T2 signal in-
tensity revealed a concentration-dependent darkening effect and
decreased significantly with increasing Fe concentration. In addi-
tion, the T2 relaxation rate (r2 ¼ 1/T2) of the MMSN-NH-Pt-CD/AD-
RGD was calculated to be 136.6 mM1S1 (Fig. 1G), suggesting the
feasibility of applying our designed MMSNs as a contrast agent in
MR imaging.
To precisely control the drug release intracellularly, the
reduction-sensitive platinum(IV) prodrug was employed as the
linker to connect the gatekeeper and mesoporous silica shell. Here,
ascorbic acid was selected as a reduction agent to trigger the release
of drug because it abound in cells and has been demonstrated to be
one of the major substance for the reduction of platinum(IV)
[46,47]. As shown in Fig. 1H, at physiological condition (PBS buffer,
pH 7.4), a negligible amount of DOX was leaked from MMSN-NH-
Pt-CD/AD-RGD, indicating that perfect capping efficiency of b-CD-

Fig. 2. In vitro tumor-targeted drug delivery confirmed by confocal laser scanning microscopy (CLSM) observations. CLSM images of HeLa cancer cells incubated with DOX loaded
MMSN-NH-Pt-CD/AD-RGD without (A) and with (B) an external magnetic field for 1 h. CLSM images of HeLa cancer cells incubated with DOX loaded MMSN-NH-Pt-CD/AD-RGD
without (C) and with (D) an external magnetic field for 2 h. CLSM images of COS7 normal cells incubated with DOX loaded MMSN-NH-Pt-CD/AD-RGD without (E) and with (F) an
external magnetic field for 1 h. CLSM images of COS7 normal cells incubated with DOX loaded MMSN-NH-Pt-CD/AD-RGD without (G) and with (H) an external magnetic field for
2 h. The concentration of DOX in each formulation was 2 mg/mL. M represented the magnetic field.
94 W.-H. Chen et al. / Biomaterials 76 (2016) 87e101

based machine. However, when the drug loaded MMSN-NH-Pt-CD/
AD-RGD was incubated with 1 mM and 10 mM ascorbic acid (pH
7.4) in PBS buffer, DOX was rapidly released. This was attributed to
ascorbic acid induced the cleavage of reduction-sensitive linker in
the progress of reducing platinum(IV) prodrug to the activated
platinum(II) drug, leading to the removal of b-CD gatekeeper and
triggering the in situ drug release. Above results confirmed the
successful construction of multifunctional MMSNs, which exhibi-
ted uniform nanostructure with suitable surface charge as well as
controlled drug release.
targeting RGD motif can enhance the uptake of particles through
specifically binding with cancer cells with avb3 integrin overex-
pressed [49,50]. With the aid of an external magnetic field,
MMSNeNHePt-CD/AD-RGD further enhanced the internalization
capability via directionally increasing RGDintegrin interactions. In
contrast, few particles were uptaken by COS7 normal cells due to
the lack of integrin expression. These results suggested that MMSN-
NH-Pt-CD/AD-RGD can targetedly deliver anticancer drug to cancer
cells instead of normal cells, which showed great promising for
selectively killing of cancer cells.

3.2. In vitro tumor-targeted drug delivery of MMSN-NH-Pt-CD/AD- 3.3. Quantitative analysis of cellular uptake and intracellular
RGD redox-triggered cytotoxicity of multifunctional MMSNs

To confirm the active targeting specificity and magnetically
enhanced drug delivery, we incubated DOX loaded MMSN-NH-Pt-
CD/AD-RGD with a cervix carcinoma cell line (HeLa cells, in which
the avb3 integrin is overexpressed) and a normal kidney fibroblast
cell line (COS7 cells, in which the avb3 integrin expression is
negative) with or without magnetic attraction. As shown in Fig. 2,
red fluorescence of DOX could be clearly observed in HeLa cancer
cells and the fluorescence intensity increased significantly with the
time extending (Fig. 2C compared with Fig. 2A). Moreover, with the
aid of magnetic field, HeLa cancer cells incubated with MMSN-NH-
Pt-CD/AD-RGD showed remarkably enhanced DOX uptake in
comparing to cells without magnetic attraction. However, only
weak red fluorescence of DOX could be detected in normal COS7
cells (Fig. 2E and G) and there was a slight increase in the presence
of magnetic field (Fig. 2F and H). This was due to the fact that active
To further quantitatively evaluate cellular uptake of multifunc-
tional MMSNs and examine the intracellular drug delivery, cells
treated with DOX loaded MMSN-NH-Pt-CD or MMSN-NH-Pt-CD/
AD-RGD were measured by confocal laser scanning microscopy
(CLSM) and flow cytometry. As shown in Fig. 3A, the red fluores-
cence of DOX stained the whole HeLa cancer cells even including
cell nuclei after 4 h incubation with MMSN-NH-Pt-CD/AD-RGD,
indicating that particles were abundantly uptaken by cells and DOX
was released in the cytoplasm effectively. Obviously, RGD targeting
motif on the surface of MMSN-NH-Pt-CD/AD-RGD could facilitate
cellular uptake of nanoparticles through specific receptor-mediated
endocytosis. Afterward the platinum(IV) prodrug was reduced by
intracellular reductive agents, leading to removal of the gatekeeper
and release of the DOX. Finally, the released DOX specifically
diffused and accumulated in the nucleus of cancer cells to induce

Fig. 3. Evaluation the intracellular redox-sensitive drug release by CLSM and quantitative analysis of cellular uptake of DOX by flow cytometry. CLSM images of HeLa cancer cells
incubated with DOX loaded MMSN-NH-Pt-CD/AD-RGD for 4 h (A) or 4 h for HeLa cancer cells pre-treated with the excess of free RGD in advance (B). CLSM images of COS7 normal
cells incubated with DOX loaded MMSN-NH-Pt-CD/AD-RGD for 4 h (C). CLSM images of HeLa cancer cells (D) and COS7 normal cells (E) incubated with DOX loaded MMSN-NH-Pt-
CD for 4 h. Flow cytometry measurement of internalized DOX signals in HeLa cancer cells (F) or COS7 normal cells (G). The mean fluorescence intensity (MFI) of HeLa cancer cells (H)
and COS7 normal cells (I) correspond to flow cytometry analysis. The cells without treatment (blank line), treating with MMSN-NH-Pt-CD for 4 h (blue line), 4 h with MMSN-NH-Pt-
CD/AD-RGD (green line), and 4 h with MMSN-NH-Pt-CD/AD-RGD and pre-treated with the excess of RGD in advance (red line). The concentration of DOX in each formulation was
2 mg/mL. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
 W.-H. Chen et al. / Biomaterials 76 (2016) 87e101 95

Fig. 4. Evaluating the intracellular MMSNs by TEM observation, ICP-AES analysis and T2-weighted MR examination. (A) TEM image of HeLa cancer cells incubated with MMSN-NH-
Pt-CD/AD-RGD 12 h (B) TEM image of COS7 normal cells incubated with MMSN-NH-Pt-CD/AD-RGD 12 h. (C) Quantification of Fe levels by ICP-AES in HeLa and COS7 cells treated
with MMSN-NH-Pt-CD/AD-RGD for 2 h, 4 h, 6 h, and 8 h, respectively. The cells without treatment were used as the blank control. Statistical significance in difference was analyzed
using student's T-Test: *p < 0.1 and **p < 0.01. (D) T2-weighted MR images of HeLa cells (1  106) incubated with MMSN-NH-Pt-CD/AD-RGD at different Fe concentrations. (E) T2-
weighted MR images of COS7 cells (1  106) incubated with MMSN-NH-Pt-CD/AD-RGD at different Fe concentrations.

apoptosis and cell death. However, when the receptor-mediated
endocytosis was blocked by free RGD peptide, the internalization
of particles was drastically decreased on account of the free RGD
competition (Fig. 3B). Similarly, limited DOX was detected in COS7
normal cells due to the less expression of integrin receptors of
normal cells (Fig. 3C). As the control, MMSN-NH-Pt-CD without
RGD motif showed little uptake of both HeLa and COS7 cells (Fig. 3D
and E), which further confirmed that RGD peptide specifically
improved the cellular uptake. The same phenomenon could also be
detected from the quantitative flow cytometry analysis. The mean
fluorescence intensity (MFI) of DOX in HeLa cancer cells was 5.3-
fold than COS7 cells after incubating with MMSN-NH-Pt-CD/AD-
RGD and 8.2-fold increases compared with that treated with
MMSN-NH-Pt-CD (Fig. 3H and I). In contrast, since free DOX has no
selectivity, similar fluorescence intensities in both HeLa and COS7
cells were observed (see Fig. S9).
Moreover, TEM was utilized to monitor the distribution of
MMSN-NH-Pt-CD/AD-RGD within HeLa and COS7 cells. As shown
in Fig. 4A and B, a multitude of MMSN-NH-Pt-CD/AD-RGD could be
observed in the cytoplasm of HeLa cancer cells (red circle) and only

Fig. 5. Evaluation the anticancer ability of multifunctional MMSNs in vitro. Cytotoxicity of DOX loaded magnetic mesoporous silica nanoparticles against (A) HeLa cancer cells and
(B) COS7 normal cells in the absence or in the presence of a magnetic field for 48 h, respectively. M represented the magnetic field.
96 W.-H. Chen et al. / Biomaterials 76 (2016) 87e101

a little was detected in COS7 normal cells, which were consistent
with above CLSM observations. The intracellular Fe contents were
further examined by ICP-AES quantitative analysis (Fig. 4C). With
increased incubation time, the Fe contents enhanced remarkably in
the HeLa cancer cells, which were 1.87-fold and 2.6-fold more than
that in the COS7 normal cells for 6 h and 8 h, respectively. Simul-
taneously, it is well known that T2 signal intensity increases in a Fe
concentration-dependent manner. The more Fe content in the cell
would reveal relatively dark T2-weighted images. As shown in
Fig. 4D and E, the HeLa cancer cells treated with MMSN-NH-Pt-CD/
AD-RGD showed notably decreased signal intensity in the T2-
weighted MR images comparing to COS7 normal cells. With
increasing Fe concentration, the dark intensity of T2-weighted
images of HeLa cancer cells became more obvious. The above re-
sults verified that the MMSN-NH-Pt-CD/AD-RGD could be inter-
nalized in cancer cells preferentially and used as promising contrast
agents for tumor-targeted MRI.
Furthermore, the in vitro cancer inhibition ability of

Fig. 6. In vivo noninvasive tumor-targeted MR imaging. (A) T2-weighted MR images of HeLa tumor-bearing mice before and after intravenous injection of MMSN-NH-Pt-CD/AD-
RGD. (B) The pseudo-color images of the tumor tissues derived from Fig. A. (C) T2-weighted MR images of HeLa tumor-bearing mice before and after intravenous injection of MMSN-
NH-Pt-CD. (D) The pseudo-color images of the tumor tissues derived from Fig. C. The two tumors were inoculated in the left and right back of the hind leg region (white arrow). To
examine magnetic targeting, an external magnet was securely fixed onto the right tumor of each mouse by bandage.
 W.-H. Chen et al. / Biomaterials 76 (2016) 87e101
multifunctional MMSNs was evaluated by MTT assay. At presented
in Fig. 5A, the HeLa cell viability after incubated with DOX loaded
MMSN-NH-Pt-CD/AD-RGD in the absence of a magnetic field was
about 30%. And the cell viability further reduced to 18% in the
presence of an external magnetic field. At the same time, we also
detected that the HeLa cell viability was more than 60% with an
external magnetic field or above 80% without an external magnetic
field after treated with DOX loaded MMSN-NH-Pt-CD. With the aid
of active targeting RGD motif, MMSN-NH-Pt-CD/AD-RGD would
preferentially enter into cancer cells in compared to MMSN-NH-Pt-
CD and significantly enhance cellular uptake. Under the intracel-
lular reductive condition, the platinum(IV) prodrug between the
gatekeeper and MMSNs were cleaved, inducing the accelerated
release of loaded drug to kill cancer cells. In comparison to litera-
ture report using gold nanoparticles as drug nanocarrier [51], the
anticancer therapeutic effect of MMSN-NH-Pt-CD/AD-RGD (IC50 for
HeLa cells was 1.2 mg/mL) was notably better than that of gold
nanoparticles (IC50 for HeLa cells was 1.8 mg/mL). To further confirm
the cell selectivity of MMSNs, the normal cell viability was also
investigated. As shown in Fig. 5B, the COS7 normal cell viabilities
were >80% after treated with different formulations of MMSNs. The
low uptake of MMSNs by normal cells resulted in the less cyto-
toxicity. In addition, the unloaded MMSN-NH-Pt-CD/AD-RGD also
exhibited some cytotoxicity to HeLa cancer cells since the plati-
num(IV) prodrug could be reduced to active platinum(II) drug to
kill cells to some extent (see Fig. S10). Consequently, these results
illustrated that MMSN-NH-Pt-CD/AD-RGD with cancer targeting
showed the capability to selectively eliminate cancer cells not
normal cells, which was beneficial to destroy cancer as well as
reduce the side effects to normal tissues.
3.4. In vivo magnetic targeting enhanced MR imaging and precise
therapy
As a noninvasive imaging technique with high spatial and
temporal resolution, MRI has been recognized as powerful tool for
early detection of cancer and evaluation the therapeutic response.
To demonstrate the magnetic targeting enhanced MR imaging of
MMSNs, two tumors were inoculated in the left and right back of
the hind leg region and the tumor on the right was in presence of an
external magnetic field while the left was not. After intravenous
injection of MMSN-NH-Pt-CD/AD-RGD or MMSN-NH-Pt-CD on the
nude mice bearing HeLa tumors, T2-weighted MR images were
taken at a given time. As shown in Fig. 6, signal attenuation at the
tumor tissues could be clearly observed in both groups at 30 min
after injecting, while the signal dropping from MMSN-NH-Pt-CD/
AD-RGD treated mice was more remarkable than that adminis-
trated MMSN-NH-Pt-CD (Fig. 6B comparing to Fig. 6D). Compared
with the simple passive EPR effect of MMSN-NH-Pt-CD, MMSN-NH-
Pt-CD/AD-RGD with the active targeting effect could enhance the
accumulation at the tumor site. The tumor targeting and retention
was further increased significantly under the guide of magnetic
force, leading to the much darker signal in T2-weighted MR images
in the right tumor (Fig. 6B, the right tumor comparing to the left
tumor). Clearly, the magnetic targeting mediated EPR effect could
overcome tumor heterogeneity and enhance the efficacious accu-
mulation of MMSNs in the tumor site, which also beneficial for
tumor-targeted therapy.
Furthermore, in vivo tumor targeting efficiency of MMSNs was
evaluated. After 24 h intravenous injection of different MMSNs, the
HeLa tumor-bearing mice was sacrificed. Tumor tissues and main
organs (heart, liver, spleen, lung and kidney) for each group were
harvested for analyzing the amounts of Si by ICP-AES. As shown in
Fig. 7, with the aid of an external magnetic field, a considerable
amount of MMSN-NH-Pt-CD/AD-RGD accumulation in tumors was
97
Fig. 7. The biodistribution of different nanoparticles (Si concentration% ID/g) in various
organs of mice collected at 24 h after intravenous injection of MMSN-NH-Pt-CD,
MMSN-NH-Pt-CD/AD-RGD, and MMSN-NH-Pt-CD/AD-RGD with an external magnetic
field. The statistical significance in difference was analyzed using student's T-Test:
*p < 0.05 and **p < 0.01.
detected (ca. 13.8% ID/g), which was much higher than that of
MMSN-NH-Pt-CD and MMSN-NH-Pt-CD/AD-RGD (ca. 2.3 and 8.9%
ID/g, respectively). To investigate antitumor efficacy of MMSNs
in vivo, thirty HeLa tumor-bearing mice were randomly divided into
five groups (n ¼ 6) and treated with PBS buffer, free DOX, DOX
loaded MMSN-NH-Pt-CD, DOX loaded MMSN-NH-Pt-CD/AD-RGD
and DOX loaded MMSN-NH-Pt-CD/AD-RGD with an external
magnetic field every other day, respectively. The injection of
different formulations of DOX had the equal dosage of 2 mg/kg and
tumor volume of each mouse was measured daily. As shown in
Fig. 8A, different DOX formulations showed significant therapeutic
effects on suppressing tumor growth in comparison to that of PBS
buffer after intravenous injection. MMSN-NH-Pt-CD/AD-RGD par-
ticles had notably higher inhibition efficacy towards tumor growth
than the free DOX and MMSN-NH-Pt-CD particles (*p < 0.05),
resulting from the active targeting ability of these particles. More
importantly, a remarkably enhanced therapeutic effect in tumor
suppression for MMSN-NH-Pt-CD/AD-RGD particles with an
external magnetic field was observed (**p < 0.01), suggesting that
the magnetic attraction increased the tumor accumulation of
MMSN-NH-Pt-CD/AD-RGD and the in situ redox-sensitive drug
release was beneficial to enhance antitumor activity and achieve
precise therapy. The superior therapeutic effect of MMSN-NH-Pt-
CD/AD-RGD particles was also substantiated directly by the average
tumor weights (Fig. 8B) as well as the representative tumor images
(Fig. 8C). Furthermore, the enriching of MMSNs in tumor tissues
was visually evidenced by Prussian blue staining. As shown in
Fig. 8EeE4, the significantly increased iron in the tumor treated by
MMSN-NH-Pt-CD/AD-RGD with a magnetic field was detected,
which was much more than that in tumors treated by MMSN-NH-
Pt-CD and MMSN-NH-Pt-CD/AD-RGD without magnetic attraction.
Consistent with the results by MR imaging, Prussian blue staining
assay confirmed the targeted tumor accumulation of MMSNs,
which contributed to selectively ablating cancer and reducing
adverse effects to normal tissues.
The enhanced antitumor effect of MMSNs was further evaluated
by Hematoxylin and eosin (H&E) staining and immunohisto-
chemical labeling of terminal deoxynucleotidyl transferased dUTP
nick end labeling (TUNEL) assay for analysis of apoptosis.
Comparing to the control group (Fig. 8F), only a spot of tumor cells
showed apoptosis/necrosis when treated with free DOX (Fig. 8F1)
and MMSN-NH-Pt-CD (Fig. 8F2). Excitingly, a multitude of tumor
cells were destroyed in the MMSN-NH-Pt-CD/AD-RGD treated
groups (Fig. 8F3 and 8F4) and more serious damages were detected
98 W.-H. Chen et al. / Biomaterials 76 (2016) 87e101

Fig. 8. Evaluating the therapeutic efficacy of multifunctional MMSNs in vivo. (A) Tumor growth curves in HeLa tumor-bearing mice after intravenous injection of different for-
mulations of DOX. The dose of DOX was 2 mg/kg. (B) The average tumor weights after various treatments. (C) The representative images of the tumors after treatment. (D)
Quantification of the percentage of TUNEL positive apoptotic cells in tumors treated with different DOX formulations. The apoptotic cells in each slide were statistically analyzed by
randomly taking three areas in a large view. *p < 0.05 was determined by a Student's t-test when the group MMSN-NH-Pt-CD/AD-RGD versus the tumors treated with free DOX and
MMSN-NH-Pt-CD, and **p < 0.01 was determined by a Student's t-test when the group MMSN-NH-Pt-CD/AD-RGD with an external magnetic field versus all other treatment groups.
Histological examination of tumor slices after treated with different DOX formulations. (E) Prussian blue staining. (F) H&E staining. (G) TUNEL immunofluorescence staining. The
nuclei were labeled with DAPI, and the apoptotic cells were green. The scale bars were 50 mm. (EeG) The group treated with PBS. (E1-G1) The group treated with free DOX. (E2-G2)
The group treated with DOX loaded MMSN-NH-Pt-CD. (E3-G3) The group treated with DOX loaded MMSN-NH-Pt-CD/AD-RGD. (E4-G4) The group treated with DOX loaded MMSN-
NH-Pt-CD/AD-RGD with an external magnetic field. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
 W.-H. Chen et al. / Biomaterials 76 (2016) 87e101 99

under the guide of magnetic force than without. In addition, the
quantitative analysis of apoptotic cells in each group by TUNEL
staining assay also confirmed the above observations. As shown in
Fig. 8D and Fig. 8GeG4, the percentage of apoptotic cells in the
tumor treated by MMSN-NH-Pt-CD/AD-RGD with a magnetic field
were about 68%, which were much more than other groups (PBS:
2%, free DOX: 18.9%, MMSN-NH-Pt-CD: 15.7%, and MMSNeNHePt-
CD/AD-RGD: 41.2%). These results further confirmed that magnetic
targeting mediated EPR effect could circulate the heterogeneity of
tumor and increase the enrichment of MMSNs in tumor sites.
Subsequently, the in situ redox-sensitive drug release promoted to
increase the therapeutic index and achieve the favorable antitumor
effects. However, it is well known that free DOX without any tumor
targeting ability only inhibit tumor growth to some extent and al-
ways induce severe side effects on normal organs [52,53]. As shown
in Fig. S11, the body weights of mice treated with free DOX showed
slightly decreased due to its systemic toxicity. The H&E staining of
the liver also revealed severe hepatotoxicity in the mice treated
with free DOX (Fig. 9B). The cell nuclei shrinkage and the extensive
apoptotic liver cells were observed (red circle). In contrast, no
significant change in the body weights was detected and no
apparent abnormality of major organs in the histopathological
examination was discovered during the treatment of MMSNs
(Fig. 9). These results demonstrated that MMSNs were favorably
biocompatible and had the excellent performance in eliminating
tumor with minimal side effects.
4. Conclusion
In summary, a multifunctional theranostic drug delivery system
based on magnetic mesoporous silica nanoparticles was designed
and prepared for tumor-targeted magnetic resonance imaging and

Fig. 9. Evaluating the biocompatibility of multifunctional MMSNs in vivo. The major organs (heart, liver, spleen, kidney and lung) were stained with H&E after different treatments.
The scale bars were 50 mm. (A) The group treated with PBS. (B) The group treated with free DOX. (C) The group treated with DOX loaded MMSN-NH-Pt-CD. (D) The group treated
with DOX loaded MMSN-NH-Pt-CD/AD-RGD. (E) The group treated with DOX loaded MMSN-NH-Pt-CD/AD-RGD with an external magnetic field.
100 W.-H. Chen et al. / Biomaterials 76 (2016) 87e101
precisely controlled therapy. The multifunctional MMSN demon-
strated the ability of selective uptake by cancer cells and induction
of the intracellular redox-sensitive release of anticancer drug to
eliminate cancer cells effectively. Moreover, the multifunctional
MMSN exhibited high contrast in MRI for locating tumor in vivo and
achieved significant antitumor efficacy with minimal side effects. It
is envisioned that the combination of magnetic nanoparticle and
mesoporous silica nanoparticle have following advantages: (1)
favourable biocompatibility and biostability, (2) remarkably
improved efficacious accumulation of magnetic mesoporous silica
nanoparticles in the targeted cancer, (3) stimuli-responsive release
of therapeutic agents, (4) integrated diagnostic and therapeutic
functions. This multifunctional theranostic nanoparticle should
have great potential in the future cancer therapy.
Acknowledgments
This work was supported by the National Natural Science
Foundation of China (51125014, 51233003 and 51533006), the
Ministry of Education of China (20120141130003) and supported by
the Fundamental Research Funds for the Central Universities
(2014203020201 and 2014203020204).
Appendix A. Supplementary data
Supplementary data related to this article can be found at http://
dx.doi.org/10.1016/j.biomaterials.2015.10.053.
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