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A smart pH-responsive nano-carrier as a drug

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delivery system for the targeted delivery of ursolic
acid: suppresses cancer growth and metastasis by
modulating P53/MMP-9/PTEN/CD44 mediated
multiple signaling pathways†
Kai Jiang,‡ Ting Chi,‡ Tao Li, Guirong Zheng, Lulu Fan, Yajun Liu, Xiufen Chen,
Sijia Chen, Lee Jia and Jingwei Shao *

Received 10th March 2017,
Accepted 4th June 2017
DOI: 10.1039/c7nr01677h
rsc.li/nanoscale
Ursolic acid (UA) has been recently used as a promising anti-tumor and cancer metastatic chemo-preven-
tive agent due to its low toxicity and liver-protecting property. However, the low bioavailability and non-
specific tumor targeting restrict its further clinical application. To address the problem, a silica-based
mesoporous nanosphere (MSN) controlled-release drug delivery system (denoted UA@M-CS-FA) was
designed and successfully synthesized, and was functionalized with folic acid (FA) and pH-sensitive chito-
san (CS) for the targeted delivery of UA to folate receptor (FR) positive tumor cells. UA@M-CS-FA were
spherical with mean diameter below 150 nm, and showed about −20 mV potential. Meanwhile,
UA@M-CS-FA exhibited a pH-sensitive release manner and high cellular uptake in FR over-expressing
HeLa cancer cells. Also, in vitro cellular assays suggested that UA@M-CS-FA inhibited cancer cell growth,
invasion and migration. Mechanistically, UA@M-CS-FA induced cancer cell apoptosis and inhibited
migration via cell cycle arrest in the G0/G1 stage, regulating the PARP/Bcl-2/MMP-9/CD44/PTEN/P53.
Importantly, in vivo experiments further confirmed that UA@M-CS-FA significantly suppressed the tumor
progression and lung metastasis in tumor-bearing nude mice. Immunohistochemical analysis revealed
that UA@M-CS-FA treatment regulated CD44, a biomarker of cancer metastasis. Overall, our data demon-
strated that a CS and FA modified MSN controlled-release drug delivery system could help broaden the
usage of UA and reflect the great application potential of the UA as an anticancer or cancer metastatic
chemopreventive agent.

Introduction therapy and metastasis. Various studies have shown that UA

Ursolic acid is a type of ursane-type pentacyclic triterpenic
acid, ubiquitous in a variety of natural medicinal plants1 with
multiple pharmacological properties, including anti-inflamma-
tory,2 liver-protective,3,4 anti-atherosclerotic,5 anti-epileptic,
anti-cancer,6 anti-epileptic,7 and anti-diabetic activities.8 In
recent years, it has garnered major attention in the field of
cancer research due to its good anti-tumor activity and low tox-
icity, which especially makes it a candidate for cancer chemo-
Cancer Metastasis Alert and Prevention Center, Fujian Provincial Key Laboratory of
Cancer Metastasis Chemoprevention and Chemotherapy, Fuzhou University,
Fuzhou 350002, China. E-mail: shaojingwei@fzu.edu.cn, shaojw12@163.com;
Tel: +86-13600802402
† Electronic supplementary information (ESI) available. See DOI: 10.1039/
c7nr01677h
‡ These authors contributed equally to this work.
exhibits growth inhibition properties against various human
cancers cell lines.9–14 Our previous studies also provided
evidence that UA and its derivatives showed potential anti-
cancer and especially cancer chemo-preventive activities.15–17
However, the poor water solubility of UA causes its low bio-
availability, and ultimately restricts its potential clinical
applications.
In recent decades, nano-technology based delivery systems
have been making a significant impact on the development of
new strategies for cancer treatment. Various nano-materials, as
drug-carriers, have been widely used in nano-drugs for cancer
treatment, such as Fe3O4 18,19 and gold nanoparticles,20 nano-
gels,21 liposomes,22 dendrimers,23 polymers,24 micelles,25
nanoemulsions,26 and silica nanoparticles.27,28 Among the
above nanocarriers, mesoporous silica nanoparticles (MSNs)
have a very high degree of attention in biomedical fields,
owing to their small size, highly-ordered mesoporous struc-

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ture, large specific surface area, large pore volumes, low tox-
icity, excellent biocompatibility, good mechanical stability and
convenient chemical modification. Hence, a variety of pharma-
ceutical drugs and fluorescent dyes have been loaded into
MSNs for controlled drug release. Many studies have reported
that some components were used as the gatekeeper of MSNs
for controlled drug delivery, for example, inorganic nano-
particles (Au, Fe3O4, CdS, and ZnO), dendrimers, antibodies,
rotaxanes, poly acrylic acid, pseudorotaxanes, and
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β-cyclodextrin.28–30 In our previous work, we designed a silica-
based mesoporous nanosphere (MSN) controlled-release
prodrug delivery system (UA@MSN-UA), which could improve
the bioavailability and extend the drug release of UA.31
Chitosan is typically used in the field of pH-responsive drug
delivery owing to its strong sensitivity in near-neutral or
slightly acidic pH environments.32,33 In order to further
enhance drug molecule specific recognition by the tumor
tissues, nanocarriers generally require further modification
with targeting molecules, such as antibodies, peptides, nucleic
acids, polysaccharides, or the small molecular compound folic
acid (FA).34 According to the literature we consulted, there is
no report that aimed at realizing targeted delivery of UA by the
CS and FA functional modification of MSN.
In the present study, a smart nanocarrier consisting of
mesoporous silica nanoparticles and folic acid conjugated
chitosan (M-CS-FA) was designed and synthesized as a smart
drug delivery system for the targeted delivery of UA to FR over-
expressing cancer cells for the first time. In our strategy, UA
was encapsulated into the MSN through non-covalent inter-
actions, and then the surface of MSN was covalently conju-
gated with CS-FA through an acid-labile amide bond.
Following the nanoparticle preparation, the physicochemical
characteristics were investigated, such as the particle size, zeta
potentials, morphology and in vitro drug release. Then, the
biological properties of UA@M-CS-FA nanoparticles were
further investigated by cellular uptake, MTT assay, invasion
assay, wound healing assay, cell cycle, cell apoptosis analysis
and western blot, respectively. In addition, its anti-tumor and
anti-metastasis effects were further investigated in vivo.
Experimental section
Characterization of the nanoparticles
For the characterization of M-C and M-CS-FA, one drop of
nanoparticles was dripped on a copper grid covered with a
nitrocellulose membrane, and transmission electron
microscopy (TEM; a JEOL JEM-1200EX microscope, Japan) was
used to observe the morphology and structure of M-C and
M-CS-FA. An Atomic Force Microscope (AFM; Bruker
MultiMode8, Germany) was used to observe the morphology of
M-C and M-CS-FA. Samples for AFM were prepared following a
standard procedure. Briefly, 8 μL of a nanoparticle diluent was
dripped on pretreated new cleaved mica, and dried at room
temperature for detection. M-C, M-CS and M-CS-FA were dis-
persed in deionized water and then the size distributions and
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zeta potentials were measured by using a Malvern Zetasizer
Nano-S90 (England). The surface areas and pore-size distri-
butions of the MSNs were examined by Brunauer–Emmett–
Teller (BET) measurements. An ASAP 2020 Physisorption
Analyzer (Micromeritics Instruments Corporation, USA) was
used to determine nitrogen-adsorption–desorption isotherms
of the M-C. A multipoint BET method was used to measure the
specific surface areas of the M-C and the pore-size distri-
butions were calculated from desorption isotherms by the
Barrett–Joyner–Halenda method. The chemical structures of
the chitosan–folic acid conjugate and the nanoparticles (M-C,
M-CS and M-CS-FA) were confirmed by Fourier transform
infrared spectroscopy (FT/IR-480). The spectra were recorded
from 4000 to 500 cm−1. In addition, CS-FA was also character-
ized by 1H-NMR on a Bruker Avance-400 MHz NMR spectro-
meter (Bruker BioSpin, MA, USA) using an acetic acid/deuter-
ium oxide solution (1/3 v/v) as a solvent. Thermogravimetric
analysis (TGA) of nanoparticles was performed using a thermo-
gravimetric analyser (TGS-II, PerkinElmer).
Drug loading into nanoparticles
Typically, UA (30 mg) and M-C (10 mg) were added to 30 mL
methanol and stirred for 24 h at room temperature. Unbound
UA, adsorbed on the surface, was removed by washing twice
with a PBS solution, after centrifuging the UA-loaded M-C
from the suspension and removing the supernatant comple-
tely. The resulting UA@M-C was freeze-dried. UV–vis spec-
troscopy was used to measure the UA concentration in the
original solution and the supernatant solution at a wavelength
of 210 nm. The UA calibration curve is shown in Fig. S3.† The
encapsulation and drug-loading efficiencies were calculated
according to the following equations:
Encapsulation ð%Þ
¼ ðThe total amount of UA  the total amount of free UAÞ=
the total amount of UA  100%:
Drug loading ð%Þ
¼ ðThe total amount of UA  the total amount of free UAÞ=
ðthe total amount of M‐C þ the total amount of UAÞ  100%:
In vitro drug release response
The in vitro release experiments were carried out under simu-
lated physiological conditions (Phosphate Buffered Saline,
PBS) at different acidic environments ( pH 5.0, 6.5 and 7.4) at
37 °C to evaluate the feasibility of using M-CS-FA as a drug
delivery carrier. In brief, 5 mg of UA@M-CS-FA were suspended
in 5 mL of a freshly made phosphate buffer solution at various
pH values (5.0, 6.5 and 7.4, respectively). The solution was put
into a dialysis tube (MWCO: 12 kDa) which was directly
immersed in 50 mL incubation medium and kept at 37 °C,
with gentle shaking (100 rpm). The release medium (2 ml) was
withdrawn at predetermined time intervals (0.5, 1, 2, 4, 8, 12,
24, 48 and 72 h). Afterwards, the same volume (2 mL) of fresh
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release medium was added to the system. The amount of free
UA in the supernatant was quantified by UV–vis spectroscopy
at a wavelength of 210 nm as above. The release rate was calcu-
lated with the formula: Drug release = (Wi/Wtotal ) × 100%,
where Wi is the amount of accumulative released UA, and
Wtotal is the total amount of UA.
Cell uptake
HeLa and HepG2 cells were placed into a 24-well plate (3 × 104
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cells per well) and incubated at 37 °C with 5% CO2 for 24 h.
After being treated with FITC labeled M-CS-FA (0.1 mg ml−1)
and M-CS (0.1 mg ml−1) for 2 h, the cells were rinsed twice
with PBS buffer (2 × 100 μL) to remove the remaining nano-
particles and dead cells. After treatment with 200 μL of a paraf-
ormaldehyde (PA) solution for 30 min at 4 °C, their nuclei
were stained with DAPI for 10 min. Finally, the cells were
observed under a confocal microscope (Zeiss LSM 780, Zeiss
Co., Germany).
To confirm whether the uptake was mediated by FR, a fluo-
rescence-activated cell sorter (FACS) was used. Briefly, HeLa
cells were seeded into 12-well plates at a density of 5 × 104 cells
per well. After 24 h, cells were incubated with various nano-
particles (FITC labeled M-CS-FA and FITC labeled M-CS)
in RPMI 1640 medium. In the competition experiments,
2 mg mL−1 FA was applied and incubated with the HeLa cells
before the nanoparticles were added. After 2 h incubation, the
cells were washed three times with PBS, trypsinized and resus-
pended in 500 μL PBS. The mean fluorescence intensity (MFI)
was measured by FACS (BD FACSAria III, USA).
Cell viability assay
In vitro cytotoxicities of the blank nanocarrier, UA and UA
loaded nanoparticles against HeLa and HepG2 cell lines were
assessed by the standard MTT assay. Briefly, cells were seeded
into 96-well plates at an initial density of 80 000 cells per mL
for 0.1 mL (8 × 103 cells per well) and cultured overnight. Cells
were treated with various concentrations of nanodrugs in
RPMI 1640 with 10% FBS. After 24 h of incubation, the
medium was removed, and a medium containing 0.5 mg mL−1
MTT reagent was added for continuous incubation for 4 h.
Then, after removing the medium, 150 μL of dimethyl sulfox-
ide (DMSO) was added per well. The absorbance at 570 nm
was measured by using a Microquant plate reader (Thermo
Fisher). Meanwhile, we explored the effect of the blank nano-
carrier, UA and UA loaded nanoparticles against HeLa and
HepG2 cell lines in blood. Blood samples were drawn from cer-
vical cancer patients when they were under operation. The
study was reviewed and approved by the hospital institutional
review board (IRB). Blood was collected into Vacutainer tubes
containing the anticoagulant EDTA. In blood, HeLa and
HepG2 cells were harvested with trypsin and resuspended in
PBS. After washing with PBS, the cells were resuspended in
50 μL PBS at a concentration of 8000 cells per μL, and 0.5 mL
of the diluted blood specimens was mixed with the cells. Then
the above samples were incubated with MSN-CS-FA, UA, and
UA loaded nanodrugs for 4 h at 37 °C, followed by washing
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three times with PBS ( pH = 7.4). The collected cells were cul-
tured on 96-cell plates in 100 μL medium and incubated for
another 24 h before being determined by MTT assay.
Western blot
HeLa cells were incubated for 24 h with 20 μg mL−1 UA,
UA@M-C, UA@M-CS, and UA@M-CS-FA for exploring the
expression of P53, Bcl-2 and for 24 h with 1 μg mL−1 UA,
UA@M-C, UA@M-CS, UA@M-CS-FA for exploring the
expression of MMP-2, and CD44 protein. Cells were washed
with cold PBS and lysed with RIPA lysis buffer for ten
minutes at an ice-cold bath, and then the supernatants of cell
lysates were recovered by centrifugation at 12 000g for 5 min
under 4 °C. The concentrations of proteins were measured by
the BCA method. Sodium dodecyl sulfate polyacrylamide
gel electrophoresis (SDS-PAGE) was performed to separate
the samples, as described previously.35 The target protein
expression was quantified by the use of the Image Lab analysis
software (Bio-Rad).
Statistical analysis
All experiments were performed in triplicate and the acquired
data are presented as the mean ± SD. Statistical significance
was determined using a Student’s t-test. The differences were
considered statistically significant if *p < 0.05 and very signifi-
cant if **p < 0.01.
Results and discussion
Preparation and characterization of UA@M-CS-FA
The procedure for the synthesis and effects of UA@M-CS-FA is
shown in Fig. 1. Firstly, MSN with the functionalization of car-
boxyl (M-C) was synthesized by co-condensation. Then the
surface of M-C was conjugated with chitosan-folic acid (CS-FA)
by the amide bond.
From the TEM analysis (Fig. 2A), it could be seen that
M-C NPs were uniform spherical nanoparticles with an average
diameter of about 100 nm. In addition, the mean particle size of
M-C was measured by using dynamic light scattering (DLS). As
revealed in Fig. 2B, it presented a relatively larger diameter
(120 nm) due to the hydrated layer surrounding the particles
with a narrow particle size distribution. The consecutive modifi-
cation processes were measured by zeta potential measure-
ments. As summarized in Fig. 2C, the zeta potential of M-C,
M-CS and M-CS-FA was −29.3 mV, +17.5 mV, and −25 mV,
respectively, and it was consistent with the modification of chit-
osan (M-CS) on the negatively charged silica surface (M-C) and
the subsequent conjugation of FA on the positively charged
M-CS. In addition, Fig. S1† shows the z-average diameter, zeta-
potential and AFM images of different nanoparticles. As shown
in Fig. 2D, the results of the nitrogen adsorption–desorption
isotherms showed that the gas adsorption isotherm is in a typical
type IV isotherm loop, which indicated that M-C was character-
istic of a porous structure. The surface areas and pore sizes of
the M-C were evaluated by BET and BJH analyses, and the
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Fig. 1 Schematic illustration of UA@M-CS-FA for targeted delivery of UA guided pH-triggered chemotherapy. (A) Schematic diagram of the prepa-
ration of UA@M-CS-FA nanoparticles. (B) UA@M-CS-FA exerted anti-metastatic effect via the inhibition of cell adhesion, invasion and migration. (C)
The tumor targeting was achieved by specific interaction between FA and tumor cells, and the pH sensitive manner was triggered by the swelling of
chitosan chains ( pH < 5.5 in the endosome).
specific surface area of the M-C was 887.2 m2 g−1. Fig. 2E
revealed that M-C had a narrow pore size distribution centered
at 2.8 nm and the pore volume was 1.32 cm−3 g−1. Moreover,
Fig. 2F depicts thermogravimetric analysis (TGA) curves of
M-C, M-CS and M-CS-FA under constant N2 flow conditions.
Upon heating to 800 °C, the total weight loss of the materials
was obtained: 37.27% (H3) for M-C, 40.99% (H2) for M-CS, and
52.98% (H1) for M-CS-FA, respectively. On heating to 100 °C,
M-C, M-CS and M-CS-FA showed a weight loss of 31.83% (L3),
20.59% (L2) and 15.59% (L1), respectively, which was the
volatilization of residual moisture from the pores of the nano-
particles. The weight loss between 100 and 800 was attributed
to the decomposition of the functionalized groups, so the
grafting rate of CS and CS-FA on M-C was calculated according
to the formula: (H3 − L3)/(100 − H3) = (H2 − L2 − W2)/
(100 − H2); (H3 − L3)/(100 − H3) = (H1 − L1 − W1)/(100 − H1).
Thus, the grafting rate of CS and CS-FA on M-C was 15.34%
and 33.31%, respectively.
Chitosan–folic acid conjugate (CS-FA) was prepared by
carbodiimide (EDC) and N-hydroxysuccinimide (NHS) mediated
polymerization. Fig. 2G shows the results of the FTIR spectra of
FA, CS, and CS-FA. CS-FA was investigated through IR spectra. It
is known that there are several peaks at 1650–1450 cm−1 which
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are the characteristic absorption peaks of the carbon skeleton
of the aromatic ring. In the pure FA the absorption bands were
observed at 1600 cm−1 and 1480 cm−1; meanwhile, in the FA-CS
the absorption bands were observed at 1590 cm−1 and
1560 cm−1. Moreover, FA and FA-CS all included the –CONH–
group. The carboxylic absorption of CvO was at around
1740 cm−1, while its position could shift to lower frequencies
due to the conjugative effect when amidated. Meanwhile, it was
found that there was an absorption band at 1700 cm−1 in FA-CS
conjugates, which demonstrated the CvO group of FA and the
–NH2 group of CS conjugated into the amide. In addition, it
was observed that the absorption position of N–H shifted to
higher frequencies in CS due to an inductive effect aroused by
the CvO group. The absorption position of N–H in CS was
1600 cm−1, and in CS-FA conjugates, it shifted to 1630 cm−1.
CS-FA was also characterized by 1H NMR spectroscopy. From
the results in Fig. S2,† it could be seen that the signals were at
δ 1.57, 2.63 and 3.23–3.41 ppm, which were attributed to the res-
onance of the monosaccharide residue protons, –COCH3, –CH–
NH–, and –CH2–O–, respectively. The signals at δ 6.27–8.30 ppm
in the 1H NMR spectra of CS-FA were attributed to the reson-
ance of the folate aromatic protons.36,37 The above results indi-
cated that CS-FA was synthesized successfully. M-CS-FA was syn-
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Fig. 2 Characterization of the nanoparticles and conjugation, and in vitro release curves of UA. (A) TEM image of M-C. (B) Size distribution of M-C.
(C) Average zeta potential of different nanoparticles. (D) Nitrogen adsorption–desorption isotherms of M-C. (E) Pore size distribution of M-C. (F) TGA
curves of different nanoparticles. (G) Fourier-transform IR spectra of FA, CS, and CS-FA. (H) Fourier-transform IR spectra of M-C, M-CS and
M-CS-FA. (I) Cumulative UA (%) from the M-CS-FA drug carrier at 37 °C in a phosphate buffer solution ( pH = 5.0, 6.5 and 7.4).

thesized from the reaction between M-C and CS-FA using EDC/
NHS between the amino group of CS-FA and the carboxyl group
of M-C. The FTIR spectra of M-C, M-CS, and M-CS-FA are pre-
sented in Fig. 2H. There was a visible characteristic absorption
peak of CvO of the –COOH group in M-C at 1715 cm−1. The
absorption position of CvO of the –COOH group could shift to
lower frequencies when there appear a conjugative effect, hence
it would show a lower frequency absorption peak when the
–COOH group and –NH2 conjugated. Interestingly, there was a
characteristic absorption peak at 1643 cm−1 in M-CS, which
showed that the –COOH group in M-C and –NH2 in CS ami-
dated. Meanwhile, it was observed that there was an absorption
peak at 1700 cm−1 in M-CS-FA caused by the –COOH group con-
tained in FA. All data displayed MSN, CS and FA conjugated.
In vitro drug loading and release behavior
Ursolic acid, as a model anticancer drug, was loaded into
M-CS-FA. UV-Vis spectroscopy was performed to measure the
drug loading and encapsulation efficiency, whose efficiencies
were 21.8% and 56.7%, respectively.
The release properties of UA from M-CS-FA nanoparticles
were measured under different pH conditions ( pH 7.4, pH 5.0
and 6.5) at 37 °C. As shown in Fig. 2I, it is clearly seen that
different pH media had a strong influence on the release of UA
from M-CS-FA nanoparticles. At neutral pH 7.4, UA-loaded
M-CS-FA exhibited a very slow release rate and only 20% of the
UA molecules were released after 72 h. Relatively,
UA@M-CS-FA showed a significantly enhanced release rate at
acidic pH 5.0 and almost 75% of the accumulative release of
UA was found for UA@M-CS-FA at acidic pH 5.0.
The reasons for the rapid UA release of UA@M-CS-FA under
pH 5.0 are as follows. On the one hand, CS-FA was conjugated
on the surface of MSN through an acid-labile amide bond.31
On the other hand, chitosan, as a smart response molecule, is
responsive to external pH-stimuli.38,39 Acidic pH 5.0 makes
these chitosan chains swell and the mesopores of the
UA@M-CS-FA open, so that UA could diffuse out of the chan-

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nels of the UA@M-CS-FA. While the pH value of the drug-
loaded CS-MSN medium was adjusted to 7.4, the shielding
layers on the mesoporous surface of the MSNs were formed
due to the deprotonation and collapse of these chitosan
polymer chains, which could effectively block the loaded UA
from escaping from the channels of the UA@M-CS-FA. In view
of the fact that the tumor microenvironment is weakly
acidic,40–42 UA@M-CS-FA exhibited a fast release in pH 5.0,
which may be conducive to tumor therapy. Thus,
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UA@M-CS-FA may be a better platform for the delivery of UA
triggered by pH.
Cell targeting ability of FITC labeled M-CS-FA
To verify the ability of M-CS-FA nanoparticles for FA-mediated
targeted delivery of UA, FITC labeled M-CS-FA nanoparticles
were prepared. The green fluorescence emitted by FITC and
the blue fluorescence emitted by DAPI were captured by
LCFM. In Fig. 3, the images are shown from top to bottom as
follows: DAPI (blue), FITC labeled M-CS-FA (green) and the
overlay of the two channels. After 2 h of incubation with
samples, only the nuclei are observed in blue color in control
groups untreated with FITC labeled M-CS-FA. Obviously, in the
overlay images, the green fluorescence in the cytoplasm in
HeLa cells, produced by FITC labeled M-CS-FA, was greater
and more closely situated around the nuclei (blue) than that in
the HepG2 resulting from FITC labeled M-CS-FA. The main
reason for this was that far fewer folate receptors are expressed
Fig. 3 Confocal fluorescence images of HeLa and HepG2 cells after incubation with free medium and FITC labeled M-CS-FA (0.2 mg mL−1).
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by HepG2 cells than by HeLa cells, resulting in far smaller
extent of receptor-mediated endocytosis. As previously
reported, FR-positive cells have a much higher FA-functional
nanoparticle uptake than FR-negative cells.43
To explore whether the entry of M-CS-FA into the cells is via
receptor-mediated endocytosis, competition experiments were
performed by FACS. Before being incubated with FITC labeled
M-CS-FA, HeLa cells were pretreated with free FA. The results
suggested that the MFI of the pretreated group decreased to
one third of the untreated group (In Fig. S4†), similar to the
MFI of M-CS, which showed that the cellular uptake of
M-CS-FA was markedly inhibited. All these results revealed that
FA ligands on UA@MSN particles play an important role in
increasing cellular uptake via FA-receptor-mediated pathway.
Effects of UA@M-CS-FA on viability
The cytotoxicities of nanoparticles with and without loading of
UA in culture medium and in blood were evaluated by the
MTT assay with the FR-positive HeLa cells and FR-negative
HepG2 cells. As depicted in Fig. 4A and B, free UA and nano-
particles with loading of UA showed cytotoxicity towards both
cell lines in a concentration-dependent manner. It was worth
noting that, in HepG2 cells, the cytotoxicity of free UA was
slightly lower than that of UA loaded nanoparticles, and
UA@M-CS-FA has a similar cytotoxicity to UA@M-C, UA@M-CS
and UA@M-FA. However, UA@M-CS-FA showed higher cyto-
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Fig. 4 Cell viability of FR-overexpressing HeLa (A and C) and FR-negative HepG2 (B and D) incubated with nanoparticles and UA loaded
nanoparticles.
toxicity than UA@M-C, UA@M-CS, UA@M-FA and free UA in
all the doses tested in HeLa cells.
Fig. 4C and D showed that M-CS-FA and UA loaded nano-
particles could downregulate the cell viability in blood, while
UA did not show significant effect of cytotoxicity, compared to
HeLa cells and HepG2 cells cultured in the medium.
UA@M-CS-FA had a lower effect on the viability of HepG2
cells, which should be attributed to the very low expression of
folate receptors on HepG2 cells. In Fig. 4, it was clearly seen
that the cytotoxicity of UA@M-CS was similar to that of
UA@M-FA. In addition, M-CS-FA had little influence on the
viability of HeLa and HepG2 cells under 1 μg mL−1–200
μg mL−1 concentration. This result again confirmed the safety
of M-CS-FA. These results indicated that M-CS-FA particles can
effectively perform targeted delivery of UA to cancer cells and
enhance the anticancer activity.
Anti-metastatic effect of UA@M-CS-FA
Cancer cell metastasis is a very complex dynamic continuous
biological process consisting of multiple relatively indepen-
dent steps. Cell adhesion, invasion and migration are the key
points of metastasis.44 Many studies have shown that UA
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induced different cell apoptosis through different mecha-
nisms.10,45,46 In the present study, the anti-metastatic effect of
UA@M-CS-FA was confirmed by cell adhesion, invasion and
wound healing assays. Fig. 5A showed representative experi-
mental images of HeLa cells. From the data of the adhesion
assay in Fig. 5B, it was clearly seen that there was no signifi-
cant difference in HepG2 cells between groups, while the
adhesion rate of HeLa cells treated with UA@M-CS-FA to
HUVECs was lower than that of other groups. Invasion assays
showed that UA, UA@M-C, UA@M-CS and UA@M-CS-FA had
different inhibition of invasion on HeLa cells, compared to the
control group. It was observed that UA@M-CS-FA nanoparticles
significantly inhibit invasion. A wound healing assay was con-
ducted to explore the effect of UA@M-CS-FA nanoparticles on
HeLa cell mobility. The wound of the control group gradually
healed after 24 hours, while a significant wound remained
uncovered in the UA@M-CS-FA treated group, which showed
that the mobility of HeLa cells was effectively inhibited by
UA@M-CS-FA nanoparticles, compared to cells treated with UA
and other nanodrugs. However, it is notable that the wound of
the group treated with UA@M-CS-FA was similar to that of the
group treated with UA@M-CS in HepG2 cells. It is also seen
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Fig. 5 Effect of UA@M-CS-FA on the mobility of HeLa and HepG2 cells. (A) HeLa cell representative images of adhesion, invasion, and scratch
assays. (B) Quantitative analysis of the inhibition of the adhesion, invasion, migration of HeLa and HepG2 cells.
that FA modification of nanodrugs improves the therapeutic
effect of UA. The above results showed that treatment of
UA@M-CS-FA at low concentrations inhibited HeLa cell
migration.
Effects of UA@M-CS-FA on the cell cycle
To further study the cell death mechanisms of nano-drugs,
a cell cycle analysis was performed by flow cytometry. The data
in Fig. 6 indicated that free UA and nano-drugs (UA@M-C,
UA@M-CS, UA@M-CS-FA) blocked cell cycle progression in the
G0/G1 phase and the G2/M phase in HeLa and HepG2 cells
under 20 μg mL−1 concentration. UA, UA@M-C and UA@M-CS
showed no obvious difference between HeLa and HepG2 cells,
consistent with the MTT results. While UA@M-CS-FA showed
obvious effect on the cell cycle in HeLa cells, the cell could be
significantly arrested in the G0/G1 and G2/M phases in the
group, compared to the other drugs (UA, UA@M-C and
UA@M-CS). However, in HepG2 cells, the cell cycle distri-
bution showed no significant difference between groups.
These results further indicated that CS-modified nanoparticles
could achieve less release before reaching the tumor site.
Meanwhile, these results were in accordance with the result
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that FA-modified nanoparticles had the potential for targeted
delivery of UA into FR-positive cancer cells.47
Analysis of apoptosis by DAPI staining
To verify whether the growth inhibitory activity of UA loaded
nanoparticles was related to the induction of apoptosis,
changes in the nuclear morphological character of HeLa
cells and HepG2 cells were assayed by DAPI staining. As shown
in Fig. S5,† there was no sign of apoptosis in the control
group. Cell death features, such as cell shrinking and
detachment, were clearly detectable in HeLa cells treated
with free UA and nano-drugs, while the nuclear changes were
more obvious in UA@M-CS-FA treated HeLa cells, resulting
in insignificant nuclear condensation and cell shrinking
accompanied by a reduction in the number of nuclei.
However, in comparison with free UA, UA@M-C, UA@M-CS
and UA@M-CS-FA, the nuclei had no obvious changes in
UA@M-CS-FA treated HepG2 cells. The results indicated that
the capability of UA@M-CS-FA to induce FR over-expressing
HeLa cancer cell apoptosis was significantly stronger than the
effect of UA, UA@M-C and UA@M-CS. The above results were
consistent with cellular uptake studies and corroborated
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Fig. 6 Cell cycle analysis of HeLa and HepG2 cells after being treated with UA, UA@M-C, UA@M-CS, and UA@M-CS-FA.

Fig. 7 Western blot analysis of PARP, PTEN, P53 and Bcl-2 expressions after HeLa cells were treated with free UA, UA loaded nanoparticles at high con-
centrations for 24 h and MMP-9, CD44 expression after HeLa cells were treated with free UA, UA loaded nanoparticles at low concentrations for 24 h.

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the MTT assay, further demonstrating the role played by
FA-mediated targeting.
Effects of UA@M-CS-FA on apoptosis and metastasis related
protein expression
To verify the molecular mechanism of anti-tumor and anti-
migration induced by UA@M-CS-FA, we assessed the four
apoptosis-related protein and two migration-related protein
expressions in HeLa cells by western blot, including PARP,
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expression was observed. Notably, UA@M-CS-FA could signifi-
cantly down-regulate the levels of PARP and Bcl-2 compared
with UA and the other drugs, while the expression of PTEN
and P53 was up-regulated by the free UA and UA loaded nano-
particles. The effects of UA@M-CS on the levels of PTEN and
P53 were equivalent to UA, but better than UA@M-C.
Meanwhile, UA@M-CS-FA could significantly induce the levels
of PTEN and P53 compared with free UA and other UA loaded
nanoparticles, whereas treatment with UA@M-CS-FA at low

PTEN, P53, Bcl-2 and MMP-9, CD44. As shown in Fig. 7, when concentrations may influence the expression of migration-
HeLa cells were treated at high concentrations of UA and UA related proteins in HeLa cells, which was consistent with our
loaded nanoparticles, a decrease in PARP and Bcl-2 protein previous study.48 Similarly, Huang et al. also found that UA

Fig. 8 In vivo experiments. (A–B) Changes of tumor volume and the body weight change curve of nude mice treated with PBS, UA, UA@M-CS, and
UA@M-CS-FA. (C) Histopathological analysis of the heart, liver, spleen and lung stained with hematoxylin and eosin after treatment with PBS, UA,
UA@M-CS, and UA@M-CS-FA. (D–E) Optical images of lungs and quantitative analysis of pulmonary metastatic nodules separated from mice receiv-
ing PBS, UA, UA@M-CS, and UA@M-CS-FA. Circles indicate the lung metastases. **p < 0.01 versus the UA@M-CS group. Immunohistochemistry ana-
lysis of CD-44 expressions of lungs after treatment with PBS, UA, UA@M-CS, and UA@M-CS-FA.

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Paper
inhibits metastasis through the down-regulation of MMP-9 in
a dose-dependent manner.49
In vivo anticancer activity of UA@M-CS-FA
The encouraging in vitro results of UA@M-CS-FA nanoparticles
on HeLa cell mediated FR warranted further in vivo assays to
evaluate its antitumor. The ability of UA@M-CS-FA to inhibit
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the growth of the primary tumor was evaluated by using a
HeLa bearing tumor model. As shown in Fig. 8A, PBS or UA
showed negligible influence on the tumor growth, while the
UA@M-CS-FA group alone significantly inhibited the HeLa
tumor growth. Notably, during the administration, there was
no significant fluctuation in the body weights of mice among
the groups (in Fig. 8B). In addition, the data in Fig. 8C indi-
cated that there were no significant differences in the histo-
logical findings between the control and various treatment
groups, indicating that M-CS-FA showed good biocompatibility
and UA@M-CS-FA treatment was safe in mice at therapeutic
doses, which was essential for their further clinical translation.
The cell apoptosis of tumor sections was evaluated through
TUNEL staining to investigate the anti-tumor activity from
the cellular level (Fig. 8C). The PBS group had almost no cell
apoptosis with nuclei, while the UA and UA@M-CS groups
exhibited slight apoptosis with a very small part of the nuclei
stained brown by TUNEL. For the UA@M-CS-FA group, most
nuclei of tumors showed apoptosis. The selective uptake in
tumor cells of UA is a very important plus factor for improving
the anti-tumor efficacy. Given the above results, UA@M-CS-FA
has the potential to be an antitumor drug for further clinical
studies.
In vivo anti-metastasis activity of UA@M-CS-FA
Finally, we evaluated the in vivo anti-metastasis activity of
UA@M-CS-FA on lung metastatic nodules and the expression
of CD44 in a mouse model of established pulmonary meta-
stasis. It is worth noting that the images of the lung tissues in
Fig. 8D and E exhibit that treatment with PBS displayed severe
lung metastasis, while UA showed a little anti-metastasis.
However, UA@M-CS-FA significantly suppressed the pulmon-
ary metastasis. Furthermore, we checked the expression of
CD44, and IHC staining findings indicated that tumors treated
with UA@M-CS-FA significantly inhibited the expression of
CD44 in HeLa tumor cells, superior to other groups, which
was consistent with the western-blot results. Surprisingly, the
effects of UA@M-CS in vivo were better than those in vitro; it
may be delivered to the tumor tissue based on the enhanced
permeability and retention (EPR) effect. So, it could inhibit
the growth and metastasis of tumors to some extent. All
these results suggest that the high loading efficiency of
UA in M-CS-FA and FA mediated UA delivery enhanced
anti-metastasis activities. Hence, these nano-drugs are found
to be safe and effective therapeutic modalities for tumor
treatment.
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Conclusion
In this study, UA@M-CS-FA has been successfully developed
for targeted delivery of UA to tumor cells and guided by pH-
triggered drug release. The CS-FA on the surface of MSNs
could swell triggered by pH and especially identify FR over-
expressing cancer cells. The nanoparticles had the attractive
feature of a high drug-loading capacity. Furthermore, M-CS-FA
was biocompatible and UA@M-CS-FA exhibited more signifi-
cant cell proliferation inhibitory action on HeLa cells com-
pared with UA@M-C, UA@M-CS and free UA alone.
Meanwhile, cell adhesion, invasion and scratch assays indi-
cated that UA@M-CS-FA at non-cytotoxic concentrations sig-
nificantly inhibited HeLa cells adhesion, invasion and immi-
gration. A western blot assay indicated that UA@M-CS-FA
induced cancer cell apoptosis and inhibited migration via reg-
ulating the P53/MMP-9/PTEN/CD44 protein expression. What’s
more, in vivo experiment further confirmed that FA mediated
delivery of UA exhibited effective anti-tumor and anti-meta-
stasis activities. These findings indicated that M-CS-FA, as a
carrier, can efficiently delivery UA to folate receptor positive
cancer cells to improve tumor therapy of UA.
Conflict of interest
The authors declare that they have no conflict of interest.
Acknowledgements
This project was supported by the National Science
Foundation of China (no. 81472767, 81673698, and 81201709).
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