Research Article

www.acsami.org

Controllable Synthesis of a Smart Multifunctional Nanoscale

Metal−Organic Framework for Magnetic Resonance/Optical
Imaging and Targeted Drug Delivery
Xuechuan Gao,† Manjue Zhai,‡ Weihua Guan,† Jingjuan Liu,† Zhiliang Liu,*,†
and Alatangaole Damirin*,‡
†
College of Chemistry and Chemical Engineering, Inner Mongolia University, Hohhot 010021, P. R. China
‡
College of Life Sciences, Inner Mongolia University, Hohhot 010021, P. R. China
*
S Supporting Information
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ABSTRACT: As a result of their extraordinarily large surfaces and well-

defined pores, the design of a multifunctional metal−organic framework
(MOF) is crucial for drug delivery but has rarely been reported. In this
paper, a novel drug delivery system (DDS) based on nanoscale MOF was
developed for use in cancer diagnosis and therapy. This MOF-based tumor
targeting DDS was fabricated by a simple postsynthetic surface modifica-
tion process. First, magnetic mesoporous nanomaterial Fe-MIL-53-NH2
was used for encapsulating the drug and served as a magnetic resonance
contrast agent. Moreover, the Fe-MIL-53-NH2 nanomaterial exhibited a
high loading capacity for the model anticancer drug 5-fluorouracil (5-FU).
Subsequently, the fluorescence imaging agent 5-carboxyfluorescein (5-FAM)
and the targeting reagent folic acid (FA) were conjugated to the 5-FU-loaded
Fe-MIL-53-NH2, resulting in the advanced DDS Fe-MIL-53-NH2-FA-
5-FAM/5-FU. Owing to the multifunctional surface modification, the obtained DDS Fe-MIL-53-NH2-FA-5-FAM/5-FU shows
good biocompatibility, tumor enhanced cellular uptake, strong cancer cell growth inhibitory effect, excellent fluorescence
imaging, and outstanding magnetic resonance imaging capability. Taken together, this study integrates diagnostic and treatment
aspects into a single platform by a simple and efficient strategy, aiming for facilitating new possibilities for MOF use for
multifunctional drug delivery.
KEYWORDS: nanoscale MOFs, fluorescence imaging, magnetic resonance imaging, targeted drug delivery, multifunctional MOFs

1. INTRODUCTION been reported often,12−16 the preparation methods are usually

Different from most of the existing pure organic and inorganic
carrier materials, metal−organic frameworks (MOFs) as a new
class of porous crystalline materials have high stability, high
surface area, large pore volume, regular porosity, and an
intriguing variety of architectures1−4 and have already emerged
as promising drug delivery platforms.5,6 In addition, the variable
pore size of MOFs can be achieved by altering the metal or
ligand, and the appeared active sites within the framework
permit an easier transmission of guest molecules,7,8 offering
unexpected advantages in the areas of drug delivery. Starting
from Fe-based MOFs as drug delivery carriers demonstrated by
Férey and co-workers,9 inspiring efforts have been committed
to exploring these fields, and several studies have been carried
out. Anionic Zn-based MOF10 was being used as a carrier of
cationic drugs, and a biocompatible porous Mg-based MOF11
was being used as an antioxidant carrier. While these MOFs
exhibit ideal drug loading and excellent drug delivery ability, the
lack of tumor-targeting capability results in strong toxicity to
normal cells, which is adverse to the drug efficacy and limits
their biomedical applications. Although nanomaterials integrat-
ing magnetic or fluorescence imaging and drug delivery have
complex, and few attempts have been made to incorporate three
functionalities: magnetic/fluorescence imaging, cell-targeting,
and drug storage delivery in one single platform based on MOF.
Considering the above-mentioned disadvantages, designing a
targeted drug delivery system (DDS) based on MOF to reduce
the secondary action of drugs during clinical treatment and
integrating multimodal imaging capabilities is a long-standing
challenge in drug development. It is generally known that iron
is not only a widespread element in nature but also an essen-
tial element in the human body, which makes it extremely
appealing for the application of Fe-MOFs-NH2 in diverse
biomedical applications.17,18 With fascinating properties such as
well-defined pores and low toxicity, nanoscale material Fe-MIL-
53-NH2 is an ideal candidate for drug carriers.19,20 Further, the
Fe-MIL-53-NH2 materials already reported show intense mag-
netism, which enables them to be effective magnetic resonance
imaging (MRI) contrast agents.21 Furthermore, the abundant
Received: November 17, 2016
Accepted: January 12, 2017
Published: January 12, 2017

© 2017 American Chemical Society 3455 DOI: 10.1021/acsami.6b14795

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ACS Applied Materials & Interfaces Research Article

amino groups on the external surface of Fe-MOFs-NH2 nano-

crystals also offer the possibility to functionalize the Fe-based
MOF for targeted imaging or therapy, which would have a great
significance in further developments. Because the external car-
boxyl on 5-carboxylfluorescein (5-FAM, Figure S1a) can be
covalently connected to the NH2 groups on the surface of
Fe-MOFs-NH2 nanocrystals, 5-FAM is appropriate as a fluo-
rescent probe for observing the drug delivery process and real-
time imaging in cells.22
To reduce an anticancer drug’s toxicity to normal cells and
enhance anticancer effects to cancer cells, targeted molecules
should be combined with the drug carrier. As we know, folic
acid (FA, Figure S1b) receptors are usually overexpressed on
the outside surface of a number of cancer cells, and FA has
excellent affinity with folate receptors (FR), leading to a selec-
tive uptake within FR-positive cancer cells and avoiding uptake
by normal tissues that do not express FR.23,24 Thus, FA is
widely chosen as a model targeting reagent to study the effi-
ciency of delivering drugs into tumor cells.25,26 Additionally, the
stability and compatibility in physiological conditions facilitate
the employment of FA as an ideal targeting molecule for
delivery systems.
5-Fluorouracil (5-FU), a pyrimidine analogue (Figure S1c),
has been one of the major clinically applied anticancer agents
for the treatment of a wide variety of tumors because it can be
incorporated into DNA and RNA, leading to cytotoxicity and
cancer cell death.27,28 Additionally, the small molecular weight
of 5-FU boosts the burgeoning biomedical applications of 5-FU Figure 1. 2D plane view of Fe-MIL-53-NH2 nanocrystalline (A and B),
as a model anticancer drug. schematic of DDS Fe-MIL-53-NH2−FA-5-FAM/5-FU (C), and
On the basis of the above background, a multifunctional schematic illustrations of DDS Fe-MIL-53-NH2-FA-5-FAM/5-FU for
MOF-based tumor targeting DDS (Fe-MIL-53-NH2-FA-5- targeting drug delivery (D).
FAM/5-FU), shown in Figure 1, was developed in the present
study. In this composite, Fe-MIL-53-NH2 is used for encap-
of the samples was obtained under a N2 atmosphere and carried out
sulating the drug and magnetic resonance imaging, whereas FA
using a NETZSCH STA449F3 thermal analyzer in the temperature
serves as the targeted reagent, and 5-FAM is employed for fluo- range from 40 to 600 °C with a heating rate was 10 °C/min. A laser
rescent imaging. Fe-MIL-53-NH2-FA-5-FAM/5-FU, obtained scanning confocal microscope (OLYMPUS, IX81) was used to con-
via a simple postmodification method, displays satisfying mag- firm the cell targeting process and cell imaging capacity. UV−vis
netic performance and excellent green fluorescence emis- spectra of the samples were recorded on a TU-1901 diode UV−visible
sion. Meanwhile, Fe-MIL-53-NH2-FA-5-FAM/5-FU can target spectrophotometer.
MGC-803 cells and exhibits significant cell growth inhibitory 2.2. Synthesis of Fe-MIL-53-NH2. Nanocrystalline Fe-MIL-53-
effects on MGC-803 cells due to the sustained release of 5-FU. NH2 was prepared by a facile low-temperature synthesis route via the
All results demonstrate that the developed DDS Fe-MIL- reaction of FeCl3·6H2O and 2-amino terephthalic acid (NH2-H2BDC)
in the ethanol solution.29 Typically, 0.1 mmol FeCl3·6H2O, 0.1 mmol
53-NH2-FA-5-FAM/5-FU possesses great potential as a novel
NH2-H2BDC, and 9 mL of ethanol solution were mixed into a three-
theranostic agent for simultaneous fluorescence/magnetic neck round-bottom flask (25 mL), transferred into a 40 °C oil bath,
resonance imaging and drug delivery in biomedicine. We and stirred for 2 h. The obtained reaction product was purified three
envision that this new hybrid Fe-MIL-53-NH2-FA-5-FAM/ times with ethanol and dried at 30 °C in a vacuum oven.
5-FU nanocomposite will be proven to be a novel drug delivery To investigate the effect of reactant concentration on the crystal
platform for cancer disease therapy in the near future. size, the amounts of FeCl3·6H2O and NH2-H2BDC were tuned from
0.1 to 0.4 mmol.
2.3. Preparation of 5-FU-Loaded Fe-MIL-53-NH2. A pure and
2. EXPERIMENTAL SECTION dried sample of Fe-MIL-53-NH2 (0.1 g) was immersed in 50 mL of a
2.1. Material and Methods. In this work, all of the starting water solution containing 5-FU (0.1 g) for 48 h. After being cen-
analytical grade reagents and solvents were acquired from commercial trifuged, the obtained product was washed with water several times.
sources and utilized without further purification. A PANalytical empy- The 5-FU loading efficiency on Fe-MIL-53-NH2 can be inferred from
rean sharp shadow system X-ray diffractometer was used to obtain the formula as follows: loading efficiency (%) = (m1 − m2)/m. In this
X-ray diffraction (XRD) patterns of the samples over the 2θ range of equation, m1 represents the initial weight of 5-FU; m2 refers to the
5−80°, and Cu Kα radiation (λ = 1.540598 Å) was used during the weight of 5-FU present in the excess of solvent, and m refers to the
testing procedure. Scanning electron microscope (SEM) images with total weight of the solid obtained. After 2 days of soaking, the loading
high quality of all samples were acquired using a HITACHI S-4800 efficiency of 5-FU on Fe-MIL-53-NH2 was 28% according to the
scanning electron microscope, and the accelerating voltage in the standard 5-FU concentration curve shown in Figure S2.
process of testing is 200 kV. A NEXUS-670 Fourier transform infrared 2.4. Synthesis of Fe-MIL-53-NH2-FA-5-FAM/5-FU. At room
spectrophotometer was utilized to assess the functional groups in temperature, 0.1 g of 5-FU-loaded Fe-MIL-53-NH2, 0.2 g of FA, and
all samples, and photoluminescence spectra of fluorescent reagents 0.2 g of 5-FAM were added to 50 mL of saturated aqueous solution of
and the obtained sample were characterized using a Hitachi F-7000 5-FU. After the solution was mixed uniformly, 0.1 g of N-(3-(dimeth-
fluorescence spectrophotometer. Thermogravimetric analysis (TGA) ylamino)propyl)-N-ethylcarbodiimide hydrochloride (EDC) as a

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dehydrating agent was added to the above mixture, which was sub- recorded at 265 nm. The cumulative release profiles were expressed
sequently stirred for 16 h in the dark. At the end of the reaction, the by cumulative release percentages of 5-FU from Fe-MIL-53-NH2-FA-
obtained Fe-MIL-53-NH2-FA-5-FAM/5-FU nanocomposite was iso- 5-FAM/5-FU as a function of time, in which the cumulative release
lated from the solution through centrifugation, followed by washing percentages of 5-FU from Fe-MIL-53-NH2-FA-5-FAM/5-FU were
with water and drying under vacuum at 25 °C. Through use of a calculated as follows: cumulative 5-FU release = amount of released
similar method to calculate loading efficiency of 5-FU on Fe-MIL- 5-FU/amount of total 5-FU × 100%.
53-NH2, the loading efficiency of 5-FU on Fe-MIL-53-NH2-FA-
5-FAM/5-FU nanocomposite was calculated to be 23%, and the
loading amounts of FA and 5-FAM on Fe-MIL-53-NH2-FA-5-FAM/ 3. RESULTS AND DISCUSSION
5-FU nanocomposite were 2.6 and 1.5%, respectively. Additionally, 3.1. Fabrication and Characterization of Fe-MIL-53-
the standard concentration curves of FA and 5-FAM are shown in NH2-FA-5-FAM/5-FU. Different reactant concentrations were
Figures S3 and S4.
applied to control the size of Fe-MIL-53-NH2, and the results
The aforementioned synthetic procedure was subsequently used to
synthesize Fe-MIL-53-NH2-FA-5-FAM and Fe-MIL-53-NH2-5-FAM/ are shown in Figure 2. Figures 2A−D show the SEM images of
5-FU. Details of the procedure are provided in the Supporting
Information.
2.5. Cytotoxicity Study. MGC-803 and HASMC cells were used
to evaluate the in vitro cytotoxicities and antitumor efficiencies of
Fe-MIL-53-NH2-FA-5-FAM, Fe-MIL-53-NH2-FA-5-FAM/5-FU, and
5-FU via the MTT method. In short, MGC-803 and HASMC cells
were seeded onto 96-well plates with a culture density of 104 cells per
well. After MGC-803 and HASMC cells were incubated for 24 h, the
initial culture medium was removed and replaced by the dispersions
of Fe-MIL-53-NH2-FA-5-FAM, Fe-MIL-53-NH2-FA-5-FAM/5-FU,
and 5-FU in various concentrations (0, 6.25, 12.5, 25, 50, 100, and
200 μg/mL). After being incubated for another 24 h, 20 μL of MTT
solution was added to each well in which the previous culture medium
has been removed and incubated for another 4 h. At last, all media
were removed from each well before 100 μL of DMSO was added, and
the cell viability was obtained by monitoring the absorbance value of
each sample at 570 nm using a microplate reader. The cell viability was
expressed as a percentage of the absorbance value of the sample well
compared to that of the control cell. All experiments were executed in
triplicate, and the results were averaged.
2.6. Cellular Uptake Study. The in vitro targeting ability of the
FA to FR was evaluated by luminescence imaging in MGC-803 and
HASMC cells. The cells were attached onto a 6-well plate for 24 h.
Afterward, the MGC-803 cells were treated with Fe-MIL-53-NH2-FA- Figure 2. SEM images of nanocrystalline Fe-MIL-53-NH2 with dif-
5-FAM/5-FU and Fe-MIL-53-NH2-5-FAM/5-FU for 4 h, and ferent reactant concentrations (A−D).
HASMC cells were incubated with Fe-MIL-53-NH2-FA-5-FAM/
5-FU for 4 h (concentration was 0.1 μg/mL). Before being observed
using a laser confocal microscope (OLYMPUS, IX81) under an excita-
tion wavelength of 473 nm, the MGC-803 and HASMC cells were
washed several times with phosphate buffered saline (PBS).
2.7. In Vivo Magnetic Resonance Imaging. All animal
experiments were conducted according to the guidelines approved
by the Institutional Animal Care and Use Committee of Peking Uni-
versity. To carry out the in vivo imaging study, athymic nude mice
bearing glioblastoma were injected with Fe-MIL-53-NH2-FA-5-FAM/
5-FU aqueous solution (5 mg/kg). At 1 h after intratumoral injection,
magnetic resonance imaging was taken by a Siemens Prisma 3.0 T MR
scanner (Erlangen, Germany) with a gradient strength up to 80 mT/m.
2.8. In Vivo Biodistribution in Different Organs. To investigate
the biodistribution of Fe-MIL-53-NH2-FA-5-FAM/5-FU in different
organs, athymic nude mice were administered the solutions via
intraperitoneal injection (10 mg/kg) and were sacrificed at the 24 h
time point postinjection. The major organs were collected, and the
fluorescence signal intensity was recorded on an IVIS imaging system Figure 3. XRD patterns of simulated Fe-MIL-53-NH2 (A) and Fe-MIL-
(Iumina II) at an excitation wavelength of 495 nm. After auto- 53-NH2 nanocrystallines prepared with different reactant concentrations
fluorescence was deducted, the biodistribution of different organs was (B−E).
concluded as the ratio of average fluorescence intensity of the indi-
vidual organ to the average fluorescence intensity of all measured the nanocrystalline Fe-MIL-53-NH2 prepared by employing
organs. decreasing amounts of FeCl3·6H2O from 0.4 to 0.1 mmol and
2.9. In Vitro Drug Release Study. The sustained 5-FU release
BDC-NH2 from 0.4 to 0.1 mmol. Interestingly, as the reactant
assays were carried out by soaking the samples in PBS (pH 7.4 and 5).
At 37 °C, a dialysis containing 0.05 g of Fe-MIL-53-NH2-FA-5-FAM/ concentrations decrease, the sizes of the nanoparticles change
5-FU was put into a 10 mL PBS solution in a 50 mL centrifuge tube. from 120 nm to 1 μm. Thus, tuning the amount of the reactant
For real-time monitoring of the release behavior of 5-FU, 2 mL of is a facile and robust way to control the particle size. To check
solution was withdrawn from the centrifuge tube at appropriate time the crystallinity, the materials were further investigated by
intervals, and the UV−vis absorption values of the solutions were XRD, which is depicted in Figure 3. The XRD reflections of all
3457 DOI: 10.1021/acsami.6b14795
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Figure 4. (a) FTIR spectra of nanocrystalline Fe-MIL-53-NH2 (A), FA (B), 5-FAM (C), and nanocrystalline Fe-MIL-53-NH2-FA-5-FAM (D); and
(b) FTIR spectra of 5-FU (A), Fe-MIL-53-NH2-FA-5-FAM/5-FU nanocomposites (B), and Fe-MIL-53-NH2-FA-5-FAM nanocomposites (C).
Figure 5. Relaxation rate 1/T2 versus concentrations of DDS Fe-MIL-
53-NH2-FA-5-FAM/5-FU using a 3T MRI scanner. T2-weighted MRI
of DDS Fe-MIL-53-NH2-FA-5-FAM/5-FU with diverse Fe concen-
trations in vitro (the inset).
Figure 6. Excitation (A) and emission (B) spectra of 5-FAM, and the
excitation (C) and emission (D) spectra of DDS Fe-MIL-53-NH2-FA-
5-FAM/5-FU.
the obtained Fe-MIL-53-NH2 nanocrystallines correspond well
with the simulated pattern, readily indexing high crystallinity
of the obtained Fe-MIL-53-NH2 nanomaterials and the same
crystal structures of all the obtained nanocrystalline Fe-MIL-
53-NH2. Due to the abundant NH2 on the surface of nano-
crystalline Fe-MIL-53-NH2 and its small particle size, the
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Fe-MIL-53-NH2 size of about 120 nm was chosen to form the
multifunctional drug carriers by conjugation with the targeted
agent (FA) an fluorescent reagent (5-FAM).
To identify the successful synthesis of Fe-MIL-53-NH2-FA-
5-FAM nanocomposites, FTIR spectra of Fe-MIL-53-NH2
(Figure 4aA), FA (Figure 4aB), 5-FAM (Figure 4aC), and
Fe-MIL-53-NH2-FA-5-FAM (Figure 4aD) were recorded.
Fe-MIL-53-NH2 exhibits the N−H symmetrical deformation
mode at 1573 cm−1, while the stretching vibrations of the
CO of carboxyl groups in FA and 5-FAM appear at 1696 and
1693 cm−1, respectively. For comparison, the FTIR spectrum of
Fe-MIL-53-NH2-FA-5-FAM shows typical bands of Fe-MIL-53-
NH2, FA, and 5-FAM, as revealed by Figure 4aD. However,
no band around 1696 cm−1 that is representative of CO of
carboxyl groups is observed in Figure 4aD, and the character-
istic peak of CO of amide groups appears at 1596 cm−1,
suggesting the successful conjugation of FA and 5-FAM to
NH2 on the surface of Fe-MIL-53-NH2. Further, confocal laser
scanning microscopy images of Fe-MIL-53-NH2-FA-5-FAM
(Figure S5) show a strong green luminescent signal, originated
from the representative fluorescence of 5-FAM, confirming the
successful conjugation of 5-FAM.
After being loaded with 5-FU, the FTIR spectrum of DDS
Fe-MIL-53-NH2-FA-5-FAM/5-FU was collected. The FTIR
spectra further indicate the presence of 5-FU on the DDS
(Figure 4b). The peak at 1655 cm−1 belongs to the vibrations
of CO in 5-FU (Figure 4bA). The broad band at 1596 cm−1
is attributed to CO of amide groups in the Fe-MIL-53-NH2-
FA-5-FAM nanomaterial (Figure 4bC). Generally, Fe-MIL-53-
NH2-FA-5-FAM/5-FU exhibits the typical absorption of 5-FU
and Fe-MIL-53-NH2-FA-5-FAM, which indicates that the
loading of 5-FU with Fe-MIL-53-NH2-FA-5-FAM was suc-
cessful. Due to the formation of DDS Fe-MIL-53-NH2-FA-
5-FAM/5-FU, the TGA curve of Fe-MIL-53-NH2-FA-5-FAM/
5-FU is extremely different from the TGA curve of Fe-MIL-
53-NH2, as shown in Figure S6. The DDS Fe-MIL-53-NH2-
FA-5-FAM/5-FU exhibits larger weight loss because of
decomposition of the loaded 5-FU, FA, and 5-FAM. Addition-
ally, postmodification and 5-FU loading cause the XRD peaks
to broaden, while the dominating peaks of the XRD spectrum
of Fe-MIL-53-NH2-FA-5-FAM/5-FU correlates with the Fe-
MIL-53-NH2 pattern, as shown in Figure S7, suggesting the
crystalline integrity of Fe-MIL-53-NH2-FA-5-FAM/5-FU.
3.2. Properties and Applications of Fe-MIL-53-NH2-
FA-5-FAM/5-FU. The transverse relaxation times (T2) of DDS
Fe-MIL-53-NH2-FA-5-FAM/5-FU with various concentra-
tions were measured, and the relaxivity values (r2) of DDS
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Figure 7. Fluorescence imaging of live MGC-803 cells cultured with Fe-MIL-53-NH2-FA-5-FAM/5-FU (A) and Fe-MIL-53-NH2-5-FAM/5-FU
(B) and HASMC cells cultured with Fe-MIL-53-NH2-FA-5-FAM/5-FU (C) for 4 h. Left, middle, and right panels are dark-field images, bright-field
images, and overlays, respectively. Scale bar: 100 μm.
Fe-MIL-53-NH2-FA-5-FAM/5-FU can be calculated by the
slope of a plot of the relaxation rate 1/T2 versus Fe concentra-
tion. As we know, dipolar interaction between the magnetic
moments of Fe-MIL-53-NH2-FA-5-FAM/5-FU and the pro-
tons in the water can cut down T2. Thus, 1/T2 increases linearly
within the tested range of Fe concentration, as revealed by
Figure 5, and DDS Fe-MIL-53-NH2-FA-5-FAM/5-FU exhibits
high relaxivity values (r2 = 18.8 mM−1 s−1) for potential clinical
use as contrast agents for T2-weighted MRI. The T2-weighted
MRI images of DDS Fe-MIL-53-NH2-FA-5-FAM/5-FU are
shown in the inset of Figure 5, and the iron concentrations in
images 2 to 7 are 0.115, 0.2308, 0.4617, 0.9235, 1.847, and
3.694 mM, respectively. It is evident that the signal intensity
decreases (darkening) with the increase in the iron
concentration, suggesting that Fe-MIL-53-NH2-FA-5-FAM/
5-FU is a better candidate for T2-weighted MRI. Furthermore,
the fluorescence testing of DDS Fe-MIL-53-NH2-FA-5-FAM/
5-FU was also performed. Clearly, compared to the fluo-
rescence spectrum of 5-FAM, the excitation and emission
spectra of Fe-MIL-53-NH2-FA-5-FAM/5-FU are similar,
suggesting that the luminescent property of Fe-MIL-53-NH2-
FA-5-FAM/5-FU is mostly derived from the 5-FAM. It is noted
that the slight discrepancies in the position of excitation and
emission wavelengths are due to the combination of 5-FAM
and Fe-MIL-53-NH2. While 5-FAM exhibits a broad emission
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band with a maximum at 530 nm (excited at 480 nm), DDS
Fe-MIL-53-NH2-FA-5-FAM/5-FU generates a broad band with
a maximum at 560 nm (excited at 495 nm), as shown in Figure 6,
which belongs to green emission and fulfills the requirements of
fluorescence imaging.
To gain more insight into the targeting ability of DDS
Fe-MIL-53-NH2-FA-5-FAM/5-FU, we carried out fluorescence
imaging tests using FA-positive MGC-803 cells and FA-negative
HASMC cells, and the results are shown in Figure 7. When
MGC-803 cells are incubated with Fe-MIL-53-NH2-FA-5-FAM/
5-FU, bright green fluorescence is observed in the cell. How-
ever, no obvious fluorescence contrast is obtained in MGC-803
cells incubated with Fe-MIL-53-NH2-5-FAM/5-FU or HASMC
cells incubated with Fe-MIL-53-NH2-FA-5-FAM/5-FU. This
observation shows that only the FA-conjugated Fe-MIL-53-
NH2-FA-5-FAM/5-FU nanocomposite shows a high affinity to
the cancer cells but has little interaction with normal cells,
confirming the targeted fluorescence imaging ability of DDS
Fe-MIL-53-NH2-FA-5-FAM/5-FU. We also investigated the
magnetic resonance imaging capability of Fe-MIL-53-NH2-FA-
5-FAM/5-FU in vivo (Figure 8). Clearly, at 1 h postinjection,
the tumor became darker, and the T2-weighted MR image
exhibits intense contrast enhancement. In brief, Fe-MIL-53-
NH2-FA-5-FAM/5-FU displays good imaging capability similar
to that of the fluorescence and magnetic resonance imaging
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Figure 8. T2-weighted MR images of tumor-bearing athymic nude
mice without Fe-MIL-53-NH2-FA-5-FAM/5-FU (A) and with
Fe-MIL-53-NH2-FA-5-FAM/5-FU via intratumoral injection (B).
Figure 9. Accumulation of Fe-MIL-53-NH2-FA-5-FAM/5-FU in major
organs excised from athymic nude mice 24 h postinjection, and ex vivo
fluorescence imaging of different organs at 24 h postinjection (inset).
systems previously reported.30−33 Further, Fe-MIL-53-NH2-
FA-5-FAM/5-FU shows stable drug loading and release
behavior, making it superior for use in practical applications.
To explore the dispersibility and stability of this DDS
Fe-MIL-53-NH2-FA-5-FAM/5-FU for in vivo application, the
biodistribution of Fe-MIL-53-NH2-FA-5-FAM/5-FU was ana-
lyzed by ex vivo fluorescence intensity of different organs 24 h
after deducting autofluorescence, and the result is shown in
Figure 9. It can be found that DDS Fe-MIL-53-NH2-FA-5-
FAM/5-FU is able to transport to tissues in vivo, and the
intensity from the liver was highest among all measured organs.
Additionally, the ex vivo fluorescence imaging of different
organs 24 h postinjection is shown in the inset of Figure 9.
Apparently, strong fluorescence signals can still be detected in
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the liver, and the XRD of Fe-MIL-53-NH2-FA-5-FAM/5-FU
after being soaked for 7 days in PBS agrees well with the
original Fe-MIL-53-NH2-FA-5-FAM/5-FU (Figure S8), sug-
gesting that this DDS Fe-MIL-53-NH2-FA-5-FAM/5-FU is
stable under physiological conditions.
It is necessary to investigate the cytotoxicity of Fe-MIL-53-
NH2-FA-5-FAM/5-FU for its potential application in bioimag-
ing. Figure 10 shows the effects of Fe-MIL-53-NH2-FA-5-FAM,
DDS Fe-MIL-53-NH2-FA-5-FAM/5-FU, and 5-FU on the
viabilities of MGC-803 and HASMC cells using the MTT
method. After MGC-803 and HASMC cells were incubated
with Fe-MIL-53-NH2-FA-5-FAM at various concentrations,
little toxicity for the nanocomposite was observed at con-
centrations up to 200 μg/mL, and the cell viability decreases
by 80%, as shown in Figures 10aA and bA, indicating that the
Fe-MIL-53-NH2-FA-5-FAM nanocomposite displays excellent
biocompatibility. Similarly, DDS Fe-MIL-53-NH2-FA-5-FAM/
5-FU and 5-FU show little toxicity to HASMC cells, and more
than 80% of cells survive at a concentration of 200 μg/mL
(Figures 10aB and aC). In contrast, incubation of DDS
Fe-MIL-53-NH2-FA-5-FAM/5-FU with MGC-803 cells exhib-
its a dose-dependent toxicity similar to that of 5-FU and
induces 45% death of MGC-803 cells when the concentration of
Fe-MIL-53-NH2-FA-5-FAM/5-FU is 200 μg/mL (Figures 10bB
and bC). These data demonstrate that DDS Fe-MIL-53-NH2-
FA-5-FAM/5-FU gives highly efficient and selective treatment
of tumor cells due to the targeting ability of FA and the release
of 5-FU.
Due to the stable and large pore size, the Fe-MIL-53-NH2
nanocomposite displays BET surface area up to 198 m2/g
(Figure S9), and the drug loading efficiency of Fe-MIL-53-
NH2-FA-5-FAM/5-FU is 23%. To validate the sustained release
property of 5-FU from the Fe-MIL-53-NH2-FA-5-FAM/5-FU
system, release experiments were carried out in PBS buffer
solutions (pH 7.4 and 5) at 37 °C, and Figure 11 shows the
drug delivery profile. Apparently, the release rates of 5-FU both
in pH 7.4 and 5 gradually decrease with time. During the first
5 h, a distinctly rapid 5-FU release from DDS Fe-MIL-53-NH2-
FA-5-FAM/5-FU was detected, which is mostly due to the fast
release of 5-FU molecules on the Fe-MIL-53-NH2-FA-5-FAM/
5-FU surface. Subsequently, drug release slows, and the gentle
release lasts for 25 h in pH 7.4, while the gentle release lasts for
20 h in pH 5. This prolonged 5-FU release is ascribed to the
extended diffusion route of 5-FU from pores and cavities of
Fe-MIL-53-NH2-FA-5-FAM/5-FU to the surrounding environ-
ment. The faster and more stable drug release in pH 5 may be
due to the slight dissolution of Fe-MIL-53-NH2 in the acidic
environment. Clearly, DDS Fe-MIL-53-NH2-FA-5-FAM/5-FU
exhibits remarkable time-release properties, making its presence
known in the area of targeted drug delivery.
4. CONCLUSION
Tumor-targeting and programmable chemotherapeutic delivery
are demonstrated by the designed DDS Fe-MIL-53-NH2-FA-5-
FAM/5-FU. This smart system provides a unique MOF plat-
form for fluorescence/magnetic resonance dual-mode imaging
and targeted drug delivery. The Fe-MIL-53-NH2-FA-5-FAM/
5-FU nanocomposite was prepared using a reflux method at
low temperature, followed by a simple postmodification method.
In vitro fluorescence imaging verifies that DDS Fe-MIL-53-NH2-
FA-5-FAM/5-FU exhibits excellent receptor-specific targeting
fluorescence imaging for FA-positive MGC-803 cells, and the
high transverse relaxivity of the nanocomposites gives it potential
DOI: 10.1021/acsami.6b14795
ACS Appl. Mater. Interfaces 2017, 9, 3455−3462
ACS Applied Materials & Interfaces
Figure 10. (a) Viabilities of HASMC cells cultured with Fe-MIL-53-NH2-FA-5-FAM (A), DDS Fe-MIL-53-NH2-FA-5-FAM/5-FU (B), and 5-FU
(C) evaluated by MTT. (b) Viabilities of MGC-803 cells cultured with Fe-MIL-53-NH2-FA-5-FAM (A), DDS Fe-MIL-53-NH2-FA-5-FAM/5-FU
(B), and 5-FU (C) evaluated by MTT.
Figure 11. Drug release profile for DDS Fe-MIL-53-NH2-FA-5-FAM/
5-FU in PBS buffer solution at pH 7.4 (A) and 5 (B).
as a contrast agent for MRI. Additionally, such DDS is also
found to generate better toxicity to cancer cells (MGC-803
cells) due to the targeted 5-FU release, and release studies show
that the 5-FU release behavior can last for more than 20 h. In
conclusion, this paper provides a brief and efficacious method
to explore MOFs as a new targeted drug delivery system which
generates significantly enhanced antitumor efficacy. These results
provide a proof of concept for a new mode of drug delivery, and
we fully expect to see this drug delivery system make its presence
known in the area of biological applications.
■
*
ASSOCIATED CONTENT
S Supporting Information
Supporting Information for this article is available on the The
Supporting Information is available free of charge on the ACS
Publications website at DOI: 10.1021/acsami.6b14795.
Chemical structure, fluorescence imaging, TGA curves,
XRD patterns, N2 adsorption−desorption isotherms, and
absorbance of 5-FU in PBS buffer solution (PDF)
■
AUTHOR INFORMATION
Corresponding Authors
*E-mail: cezlliu@imu.edu.cn.
*E-mail: bigaole@imu.edu.cn.
ORCID
Zhiliang Liu: 0000-0003-3917-6014
Notes
The authors declare no competing financial interest.
3461
Research Article
■ ACKNOWLEDGMENTS
All authors received funding from the Natural Science
Foundation of China (Grant 21361016) and the Inner
Mongolia Autonomous Region Fund for Natural Science
(Grant 2013ZD09).
■ REFERENCES
(1) Della Rocca, J.; Liu, D.; Lin, W. Nanoscale Metal−Organic
Frameworks for Biomedical Imaging and Drug Delivery. Acc. Chem.
Res. 2011, 44, 957−968.
(2) Yoon, M.; Srirambalaji, R.; Kim, K. Homochiral Metal−Organic
Frameworks for Asymmetric Heterogeneous Catalysis. Chem. Rev.
2012, 112, 1196−1231.
(3) Keskin, S.; Kizilel, S. Biomedical Applications of Metal Organic
Frameworks. Ind. Eng. Chem. Res. 2011, 50, 1799−1812.
(4) Zhang, T.; Lin, W. Metal−organic Frameworks for Artificial
Photosynthesis and Photocatalysis. Chem. Soc. Rev. 2014, 43, 5982−
5993.
(5) James, S. L. Metal-organic frameworks. Chem. Soc. Rev. 2003, 32,
276−288.
(6) McKinlay, A. C.; Morris, R. E.; Horcajada, P.; Férey, G.; Gref, R.;
Couvreur, P.; Serre, C. BioMOFs: Metal−Organic Frameworks for
Biological and Medical Applications. Angew. Chem., Int. Ed. 2010, 49,
6260−6266.
(7) Wang, C.; Liu, D.; Lin, W. Metal−Organic Frameworks as A
Tunable Platform for Designing Functional Molecular Materials. J. Am.
Chem. Soc. 2013, 135, 13222−13234.
(8) Horcajada, P.; Gref, R.; Baati, T.; Allan, P. K.; Maurin, G.;
Couvreur, P.; Férey, G.; Morris, R. E.; Serre, C. Metal−Organic
Frameworks in Biomedicine. Chem. Rev. 2012, 112, 1232−1268.
(9) Horcajada, P.; Chalati, T.; Serre, C.; Gillet, B.; Sebrie, C.; Baati,
T.; Eubank, J. F.; Heurtaux, D.; Clayette, P.; Kreuz, C. Porous Metal−
organic-framework Nanoscale Carriers as A Potential Platform for
Drug Delivery and Imaging. Nat. Mater. 2010, 9, 172−178.
(10) Wang, H. N.; Yang, G. S.; Wang, X. L.; Su, Z. M. pH-induced
Different Crystalline Behaviors in Extended Metal−organic Frame-
works based on the Same Reactants. Dalton Trans. 2013, 42, 6294−
6297.
(11) Cooper, L.; Hidalgo, T.; Gorman, M.; Lozano-Fernández, T.;
Simón-Vázquez, R.; Olivier, C.; Guillou, N.; Serre, C.; Martineau, C.;
Taulelle, F. A Biocompatible Porous Mg-gallate Metal−organic
Framework as An Antioxidant Carrier. Chem. Commun. 2015, 51,
5848−5851.
(12) Liu, K. G.; Zhu, Z.; Wang, X. Y.; Gonçalves, D.; Zhang, B.;
Hierlemann, A.; Hunziker, P. Microfluidics-based Single-step Prepara-
tion of Injection-ready Polymeric Nanosystems for Medical Imaging
and Drug Delivery. Nanoscale 2015, 7, 16983−16993.
(13) Wong, P. T.; Chen, D. X.; Tang, S. Z.; Yanik, S.; Payne, M.;
Mukherjee, J.; Coulter, A.; Tang, K.; Tao, K.; Sun, K.; Baker, J. R., Jr;
DOI: 10.1021/acsami.6b14795
ACS Appl. Mater. Interfaces 2017, 9, 3455−3462
ACS Applied Materials & Interfaces Research Article

Choi, S. K. Modular Integration of Upconverting Nanocrystal- superparamagnetic iron oxide nanoparticles. J. Mater. Chem. B 2016, 4,
Dendrimer Composites for Folate Receptor-Specific NIR Imaging 3969−3981.
and Light-Triggered Drug Release. Small 2015, 11, 6078−6090. (33) Liu, Y.; Kang, N.; Lv, J.; Zhou, Z. J.; Zhao, Q. L.; Ma, L. C.;
(14) Chen, W. H.; Luo, G. F.; Lei, Q.; Cao, F. T.; Fan, J. X.; Qiu, W. Chen, Z.; Ren, L.; Nie, L. M. Deep Photoacoustic/Luminescence/
X.; Jia, H. Z.; Hong, S.; Fang, F.; Zeng, X.; Zhuo, R. X.; Zhang, X. Z. Magnetic Resonance Multimodal Imaging in Living Subjects Using
Rational Design of Multifunctional Magnetic Mesoporous Silica High-Efficiency Upconversion Nanocomposites. Adv. Mater. 2016, 28,
Nanoparticle for Tumor-targeted Magnetic Resonance Imaging and 6411−6419.
Precise Therapy. Biomaterials 2016, 76, 87−101.
(15) Song, W. Y.; Di, W. H.; Qin, W. P. Synthesis of mesoporous-
silica-coated Gd2O3: Eu@silica particles as cell imaging and drug
delivery agents. Dalton Trans. 2016, 45, 7443−7449.
(16) Shen, B. B.; Ma, Y.; Yu, S. Y.; Ji, C. H. Smart Multifunctional
Magnetic Nanoparticle-Based Drug Delivery System for Cancer
Thermo-Chemotherapy and Intracellular Imaging. ACS Appl. Mater.
Interfaces 2016, 8, 24502−24508.
(17) Della Rocca, J.; Liu, D. M.; Lin, W. B. Nanoscale Metal−
Organic Frameworks for Biomedical Imaging and Drug Delivery. Acc.
Chem. Res. 2011, 44, 957−968.
(18) Carné, A.; Carbonell, C.; Imaz, I.; Maspoch, D. Nanoscale
Metal−organic Materials. Chem. Soc. Rev. 2011, 40, 291−305.
(19) Lin, W. B.; Rieter, W. J.; Taylor, K. M. L. Modular Synthesis of
Functional Nanoscale Coordination Polymers. Angew. Chem., Int. Ed.
2009, 48, 650−658.
(20) Spokoyny, A. M.; Kim, D.; Sumrein, A.; Mirkin, C. A. Infinite
Coordination Polymer Nano- and Microparticle Structures. Chem. Soc.
Rev. 2009, 38, 1218−1227.
(21) Serre, C.; Mellot-Draznieks, C.; Surblé, S.; Audebrand, N.;
Filinchuk, Y.; Férey, G. Role of Solvent-Host Interactions That Lead
to Very Large Swelling of Hybrid Frameworks. Science 2007, 315,
1828−1831.
(22) Zhang, P. C.; Lock, L. L.; Cheetham, A. G.; Cui, H. G.
Enhanced Cellular Entry and Efficacy of Tat Conjugates by Rational
Design of the Auxiliary Segment. Mol. Pharmaceutics 2014, 11, 964−
973.
(23) Prabaharan, M.; Grailer, J. J.; Pilla, S.; Steeber, D. A.; Gong, S.
Q. Folate-conjugated Amphiphilic Hyperbranched Block Copolymers
based on Boltorn®H40, Poly(l-lactide) and Poly(ethylene glycol) for
Tumor-targeted Drug Delivery. Biomaterials 2009, 30, 3009−3019.
(24) Sudimack, J.; Lee, R. J. Targeted Drug Delivery via the Folate
Receptor. Adv. Drug Delivery Rev. 2000, 41, 147−162.
(25) Mansoori, G. A.; Brandenburg, K. S.; Shakeri-Zadeh, A. A
Comparative Study of Two Folate-Conjugated Gold Nanoparticles for
Cancer Nanotechnology Applications. Cancers 2010, 2, 1911−1928.
(26) Stella, B.; Arpicco, S.; Peracchia, M. T.; Desmaële, D.; Hoebeke,
J.; Renoir, M.; D’Angelo, J.; Cattel, L.; Couvreur, P. Design of Folic
Acid-Conjugated Nanoparticles for Drug Targeting. J. Pharm. Sci.
2000, 89, 1452−1464.
(27) Carlsson, M.; Gustavsson, M.; Hu, G. Z.; Murén, E.; Ronne, H.
A Ham1p-Dependent Mechanism and Modulation of the Pyrimidine
Biosynthetic Pathway Can Both Confer Resistance to 5-Fluorouracil in
Yeast. PLoS One 2013, 8, e52094.
(28) Mojardín, L.; Botet, J.; Quintales, L.; Moreno, S.; Salas, M. New
Insights into the RNA-Based Mechanism of Action of the Anticancer
Drug 5′-Fluorouracil in Eukaryotic Cells. PLoS One 2013, 8, e78172.
(29) Li, Y. T.; Tang, J. L.; He, L. C.; Liu, Y.; Liu, Y. L.; Chen, C. Y.;
Tang, Z. Y. Core−Shell Upconversion Nanoparticle@Metal−Organic
Framework Nanoprobes for Luminescent/Magnetic Dual-Mode
Targeted Imaging. Adv. Mater. 2015, 27, 4075−4080.
(30) Wu, Q.; Cheng, Q. Q.; Yuan, S. M.; Qian, J. C.; Zhong, K.;
Qian, Y. F.; Liu, Z. Y. A Cell-penetrating Protein Designed for Bimodal
Fluorescence and Magnetic Resonance Imaging. Chem. Sci. 2015, 6,
6607−6613.
(31) Sun, Y. Q.; Wang, D. D.; Zhao, T. X.; Jiang, Y. N.; Zhao, Y. Q.;
Wang, C. X.; Sun, H. C.; Yang, B.; Lin, Q. Fluorescence-Magnetism
Functional EuS Nanocrystals with Controllable Morphologies for Dual
Bioimaging. ACS Appl. Mater. Interfaces 2016, 8, 33539−33545.
(32) Lam, T.; Avti, P. K.; Pouliot, P.; Tardif, J. C.; Rhéaume, E.;
Lesage, F.; Kakkar, A. Magnetic resonance imaging/fluorescence dual
modality protocol using designed phosphonate ligands coupled to

3462 DOI: 10.1021/acsami.6b14795

ACS Appl. Mater. Interfaces 2017, 9, 3455−3462