A novel pH-responsive Fe-MOF system for enhanced cancer treatment mediated by the Fenton reaction

Article abstract of DOI:10.1039/d0nj05105e

Recently, the toxic hydroxyl radical (·OH) has received wide interest for inducing cell apoptosis by increasing the intracellular reactive oxygen species (ROS) levels. Herein, a cationic polymer (MV-PAH) was rationally synthesized, and a new pH-responsive nanoreactor (DOX@Fe-MOF@PEM) was modified with the prepared cationic polymer MV-PAH multilayers by layer-by-layer (LBL) technique. The polyelectrolyte multilayer (PEM) disassembled and notably enhanced drug delivery in the weak acid environment of the tumor cell. In the cytotoxicity and apoptosis experiments, DOX@Fe-MOF@PEM revealed an advanced repression of cancer. The H₂O₂induced by DOX and MV-PAH could be catalyzed to generate hypertoxic ·OH through intracellular iron ions based on a Fenton-like reaction, which thereby improved the anticancer effect of DOX. This realistic strategy may create a potential way to provide a combination of ROS and chemotherapy to improve the anti-cancer effect.

Full text of DOI:10.1039/d0nj05105e

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A novel pH-responsive Fe-MOF system for
enhanced cancer treatment mediated by the
Fenton reaction†
Cite this: New J. Chem., 2021,
45, 3271
Senlin Wang,‡a Hongshuai Wu,‡a Kai Sun,a Jinzhong Hu,a Fanghui Chen,a Wen Liu,a
Jian Chen,a Baiwang Sun *a and Abul Monsur Showkot Hossain*b
Recently, the toxic hydroxyl radical (ꢀOH) has received wide interest for inducing cell apoptosis by
increasing the intracellular reactive oxygen species (ROS) levels. Herein, a cationic polymer (MV-PAH)
was rationally synthesized, and a new pH-responsive nanoreactor (DOX@Fe-MOF@PEM) was modified
with the prepared cationic polymer MV-PAH multilayers by layer-by-layer (LBL) technique. The
polyelectrolyte multilayer (PEM) disassembled and notably enhanced drug delivery in the weak acid
environment of the tumor cell. In the cytotoxicity and apoptosis experiments, DOX@Fe-MOF@PEM
revealed an advanced repression of cancer. The H2O2 induced by DOX and MV-PAH could be catalyzed
to generate hypertoxic ꢀOH through intracellular iron ions based on a Fenton-like reaction, which
thereby improved the anticancer effect of DOX. This realistic strategy may create a potential way to
provide a combination of ROS and chemotherapy to improve the anti-cancer effect.
Received 17th October 2020,
Accepted 7th January 2021
DOI: 10.1039/d0nj05105e
rsc.li/njc
ototoxicity and nephrotoxicity) have limited further clinical
application.19–21 The great advances in nanoscience have estab-
1. Introduction
Oxidation and antioxidants in healthy humans are in dynamic lished the tremendous latent clinical capacity of nanomaterials.22
equilibrium, and the body usually maintains suitable levels of Targeted administration can selectively transport cytotoxic drug
ROS.1,2 Breaking the threshold of ROS levels causes oxidative molecules to the target site, which is an effective method for
damage to cells, leading to apoptosis or necrosis.3–6 According to reducing systemic toxic side effects and improving the therapeutic
recent reviews, ROS play a critical role in the therapeutic effects effects.23,24 A switchable ‘‘gatekeeper’’ that intelligently controls
and side effects of chemotherapy agents.7–10 Many studies have drug release via specific stimuli can appreciably heighten the
shown that chemotherapy drugs, such as doxorubicin (DOX) and therapeutic effect of drugs.25
cisplatin, can mediate the activation of nicotinamide adenine
The polyelectrolyte pair, usually consisting of the polycation
dinucleotide phosphate (NADPH) oxidase (NOX), thereꢁby trigger- polyallylamine hydrochloride (PAH) and polystyrene sulfonate
ing the conversion of oxygen to superoxide radicals (O2 ꢂ), which (PSS), forms a type of polyelectrolyte multilayer (PEM), which
further generate H2O2 through the superoxide dismutase is gradually fabricated as a multilayered ultrathin film by layer-
enzyme.11,12 Through the Fenton reaction mediated by Fe2+/ by-layer (LBL) technology.26,27 Due to its effectivity, biocom-
Fe3+, H2O2 could be catalyzed to produce highly reactive ꢀOH, patibility and accessibility, the PEM has already received
which may cause oxidative damage to cancer cells’ lipids, widespread attention in the fields of biomaterials.22,28 Our group
proteins and DNA, thereby improving the anticancer activity of earlier developed a hollow mesoporous silica nanoparticle
chemotherapy drugs.11–14 This route also guides the way for the (HMSN@PEM) with a pH-sensitive PEM coating to realize the
development of chemotherapy drugs in the future.13,15–18
specific release of DOX and glucose oxidase in the tumor
Chemotherapy drugs play a vital role in the treatment of microenvironment for combined starvation and also drug
malignant tumors, but their serious side effects (nausea, therapy.29,30
Methyl viologen (MV or PQ) is a bipyridyl herbicide. Intriguingly,
a School of Chemistry and Chemical Engineering, Southeast University,
Nanjing 211189, P. R. China. E-mail: chmsunbw@seu.edu.cn
b School of Chemistry and Chemical Engineering, Jiangsu University, Zhenjiang,
Jiangsu 212013, P. R. China. E-mail: monsur_12@yahoo.com
† Electronic supplementary information (ESI) available. See DOI: 10.1039/
d0nj05105e
the genotoxicity and cytotoxicity of MV may be related to its ability
to generate ROS. It has already been firmly established as an
electron acceptor that acts on the redox reaction in cells to generate
a large number of active free radicals, causing lipid peroxidation of
cell membranes and oxidative damage of tissue cells.31,32 Drawing
on this idea, herein, we first confirmed the synthesis of a cationic
‡ These authors have contributed equally to the work.
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Scheme 1 A novel pH-responsive Fe-MOF system for enhancing cancer treatment mediated by a Fenton reaction.
polymer (MV-PAH), then constructed a novel pH-responsive Fe- was stirred at room temperature for 12 h. The product was
MOF system modified with MV-PAH by LBL technique, encapsulat- collected by filtration and washed with acetonitrile to obtain a
ing the chemotherapeutic DOX for enhanced cancer treatment pure yield of orange powder. Yield: 85.9% (2.56 g, 8.59 mmol).
mediated by a Fenton reaction. As shown in Scheme 1, DOX and M.p. = 247–248 1C; 1H NMR (600 MHz, DMSO) d 9.18 (d, J = 6.5
iron were released by the iron-based MOF nanoparticle in the weak Hz, 2H), 8.88 (dd, J = 4.6, 1.5 Hz, 2H), 8.65 (d, J = 6.7 Hz, 2H), 8.07
acid environment of the tumor cell. Then, H2O2 induced by DOX (dd, J = 4.5, 1.6 Hz, 2H), 4.42 (s, 3H).
ꢁ
and MV could be catalyzed to generate OH by intracellular iron
In the second step, 1-(2-carboxyethyl)-10-methyl-4,40-bipyri-
ions, resulting in enhanced antitumor activity. Such a smart drug- dinium dichloride (2) was synthesized according to the litera-
delivery nanosystem may provide a promising candidate for ele- ture with some modifications.35,36 Briefly, to a solution of
vated cancer therapy.
complex 1 (1.49 g, 5 mmol) in acetonitrile (30 mL) was added
3-bromopropionic acid (3.06 g, 20 mmol), and the reaction
mixture was heated to reflux and stirred for 24 h. The reaction
mixture was filtered and also washed with acetonitrile. The
residue was dissolved in THF (5 mL) and precipitated from
hexane (20 mL), and then dissolved in water, then NH4PF6
solution was added to replace the counter-anion with PF6ꢂ. The
2. Experimental section
2.1. Materials
3-(4,5-Dimethyl-thiazol-2-yl)-2,5-diphenyltetrazolium bromide
(MTT), poly(sodium 4-styrenesulfonate) (PSS, Mw = 70 000),
doxorubicin hydrochloride (DOX), 20,70-dichlorofluorescin dia-
cetate (DCFH-DA), 2-aminoterephthalic acid, poly(allylamine
hydrochloride) (PAH, Mw = 15 000), 4,40-bipyridine and 3-
bromopropionic acid were supplied by Sigma-Aldrich Chemical
Company (St. Louis, MO). V-FITC/PI was obtained from Beyo-
time Biotechnology (Haimen, Jiangsu, China). The rest of the
materials were commercially available.
ꢂ
PF6 salt was filtered, washed with water, and dried under
vacuum overnight. The PF6ꢂ salt was then dissolved in CH3CN,
and tetraethylammonium chloride was added to replace the
counter-anion with Clꢂ to yield a red powder, which was dried
1
in vacuo. Yield: 50% (0.32 g, 0.60 mmol). H NMR (600 MHz,
D2O) d 9.07 (d, J = 6.9 Hz, 2H), 8.95 (d, J = 6.8 Hz, 2H), 8.44 (d, J =
6.8 Hz, 2H), 8.43 (d, J = 6.8 Hz, 2H), 4.87 (t, J = 6.4 Hz, 2H), 4.40
(s, 3H), 3.04 (t, J = 6.4 Hz, 2H).
For the synthesis of MV-PAH, complex 2 (200 mg, 0.61 mmol),
EDC (128.2 mg, 0.67 mmol) and NHS (76.9 mg, 0.67 mmol) were
dissolved in 5 mL of water. Then, PAH (84 mg, 5.6 mmol)
was injected, and the purification was carried out by dialysis
(MWCO = 3000 Da) after the mixture was stirred overnight. The
product was lyophilized and analyzed using spectroscopic
techniques.
2.2. Synthesis of MV-PAH
MV-PAH was prepared by a three-step process as illustrated in
Scheme 2.
First, 1-methyl-4,40-bipyridinium iodide (1) was prepared
according to the procedure found in literature.33,34 To a solution
of 4,40-dipyridyl (1.56 g, 10 mmol) in acetonitrile (30 mL) was
added methyl iodide (1.42 g, 10 mmol), and the reaction mixture
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Scheme 2 Synthesis route of MV-PAH.
2.3. Preparation of Fe-MOF nanoparticles
To determine the DOX loading, the supernatant and the
washing solutions were measured using a Jasco V530 UV-Vis
spectrophotometer, with a standard curve method, at the
wavelength of 480 nm. The drug loading capacity (DLC) was
calculated by the following formula:
Fe-MOF nanoparticles were prepared according to the procedure
in the literature, with slight modifications.15 First, to a solution of
Pluronic F-127 (320 mg) and FeCl3ꢀ6H2O (357 mg, 1.32 mmol) in
Milli-Q water (30 mL) was added acetic acid (0.8 mL). Later, 2-
aminoterephthalic acid (120 mg, 0.66 mmol) was injected after
the mixture was stirred for an hour. Finally, the mixture was
transferred to an autoclave and heated for 20 h at 110 1C and then
the mixture was stirred for a further 1 h. The nanoparticles were
collected and washed several times using Milli-Q water and
ethanol by centrifuging at 8000 rpm for 15 minutes, yielding Fe-
MOF nanoparticles as brown powders after drying under vacuum.
Weight of drug in nanoparticles
DLC ð%Þ ¼
ꢃ 100
Weight of total nanoparticles
The Brookhaven A8530 instrument was used to determine
the size and zeta potential of the nanoparticles by dynamic light
scattering (DLS). The surface charge of the nanoparticles was
tested by a Zeta Plus zeta potential analyzer. A JEM-100CXII
transmission electron microscope was used to observe the
surface morphology of the prepared nanoparticles through a
transmission electron microscope (TEM) at 25 1C with an
acceleration voltage of 180 kV. The pore size distribution and
surface area of Fe-MOF were studied using a nitrogen (N2)
adsorption–desorption method, which was carried out on a
Brunauer–Emmett–Teller Micromeritics Tristar 3000 analyzer.
The iron mass of the particles was investigated using an ICP-MS
7400 instrument.
2.4. Preparation of DOX@Fe-MOF@PEM nanoparticles
As shown in Scheme 1, DOX@Fe-MOF@PEM (DOX@Fe-
MOF@PSS@MV-PAH@PSS) nanoparticles were formulated
according to a previous work with slight modifications.11
For DOX loading, DOX (10 mg) and Fe-MOF (20 mg) were
dispersed in PBS (20 mL), then stirred overnight at room tem-
perature in the dark. Then, DOX@Fe-MOF was collected and
washed with PBS by centrifugation (7000 rpm, 10 min). Note that
DOX concentrations in the supernatant liquid samples were
measured by UV-Vis spectroscopy using a DOX standard curve.
For polyelectrolyte multilayer coating on the surfaces of
DOX@Fe-MOF, DOX@Fe-MOF@PEM was prepared by the
2.5. Stability of DOX@Fe-MOF@PEM nanoparticles
LBL technique according to our past work.29 Briefly, PSS and The prepared DOX@Fe-MOF and DOX@Fe-MOF@PEM nano-
MV-PAH were dissolved in 0.5 M NaCl (2 mg mL-1). DOX@ particles were maintained at 4 1C for a particular time. The
Fe-MOF solution (2.0 wt%, 20 mL) was added to the negatively stability was studied by monitoring changes in nanoparticle
charged PSS solution (20 mL), and the mixture was stirred for size and PDI at particular time intervals (after 3, 7, 14, 21 and
2 h. The particles were collected by centrifugation (6000 rpm, 28 days). Furthermore, the nanoparticle suspension was
8 min), cleaned and dispersed in table salt. Then, 20 ml of centrifuged at 2000 rpm for 8 minutes to make sure that
positively charged MV–PAH was injected and stirred for another impurities were settled as planned, and then a 3 mL aliquot
2 h, centrifuged, cleaned and suspended with the table salt extracted from the supernatant was investigated. The PDI and
(2 mg mL-1 NaCl), and another PSS layer was repeated and size of nanoparticles were measured by DLS. Each sample was
alternately coated on the surface of collection.
tested in triplicate.
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2.6. In vitro pH-triggered drug release
2.9. ROS generation
The drug release of the prepared DOX@Fe-MOF and DOX@ To determine the formation of ROS, DCFH-DA, a fluorogenic
Fe-MOF@PEM was performed using dialysis bag diffusion technol- substrate that could be oxidized by ROS to the highly fluorescent
ogy. Briefly, the prepared nanoparticles were suspended in 4 mL PBS dichlorofluorescein (DCF), was selected to detect the intracellu-
(pH = 5.5 and 7.4). The suspension was then placed in a dialysis bag lar production of ROS. In short, on the day before treatment,
(MWCO = 3000 Da), and the dialysis process was observed against A549 cells were grown in a 96-well plate at a density of 2 ꢃ 105
PBS at 25 1C in the dark under constant stirring. The released mass cells per well in 200 mL of complete cell culture medium. Cells
of DOX was determined by comparing the specific absorption peak were also pretreated with various formulations for 24 h. Then,
obtained using UV-vis with the standard curve.
the culture media were replaced with PBS. After that, 200 mL of
fresh culture media containing 20 mM DCFH-DA was added to
each well for 30 min for dye loading at 37 1C. Eventually, the cells
were quantified by flow cytometry after washing twice with fresh
medium.
2.7. ꢁOH generation
Measurements were carried out according to a previous report15 with
slight modifications. Briefly, different concentrations of Fe-MOF (0, 5,
10, 20, 40, and 80 mg mLꢂ1) were added to 10 mL PBS buffer solution
(pH 5.5) containing H2O2 (1 mM) and 3,30,5,50-tetramethylbenzidine
2.10. In vitro cytotoxicity
(TMB, 0.25 mM) and maintained at 37 1C for 5 min. The absorbance The cells (A549 and MCF-7) were seeded at a density of 6 ꢃ 103
spectra were observed using UV-vis spectroscopy.
cells per well. The cells were exposed to free DOX, Fe-MOF,
DOX@Fe-MOF and DOX@Fe-MOF@PEM formulations at
different DOX concentrations (2.5, 5, 10, 20, and 40 mg mLꢂ1
)
2.8. Cellular uptake
for 24 h at 37 1C. Each well was injected with 150 mL MTT
solution (5.0 mg mlꢂ1) and also incubated for 4 h at 37 1C.
Subsequently, the medium was replaced with 150 mL DMSO to
dissolved the resultant purple crystals. Then, the plates were
shaken on the table concentrator for 15 min. Finally, the
absorption at 490 nm was measured by a microplate reader.
Confocal laser scanning microscopy (CLSM) was employed to
qualitatively measure the cell interaction with the nanoparticles
DOX@Fe-MOF and DOX@Fe-MOF@PEM. Briefly, the cells
(A549) were placed at a density of approximately 1 ꢃ 105 cells
per dish in a F20 mm confocal laser dish. After overnight
culture, the cells were incubated with DOX@Fe-MOF and
DOX@Fe-MOF@PEM nanoparticles. After 4 h incubation, the
medium was poured out, and the cells were washed with PBS
2.11. Cell apoptosis
three times. Finally, the cells were stained with DAPI and For apoptosis, A549 cells were cultured at a density of approxi-
incubated for 15 min. The intracellular distribution of nano- mately 1 ꢃ 105 cells per well for 24 h. Later, the cells were
particles was then cautiously observed with CLSM.
incubated with DOX, DOX@Fe-MOF, and DOX@Fe-MOF@PEM
Fig. 1 (A) 1H NMR spectra of complex 2, PAH and MV-PAH; (B) UV-vis spectroscopy of MV-PAH, PAH and complex 2 in aqueous solution; (C) UV
calibration curve of 1-(2-carboxyethyl)-10-methyl-4,40-bipyridinium dichloride in water at l = 257 nm.
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Fig. 2 (A) TEM image of Fe-MOF; (B) TEM image of DOX@Fe-MOF@PEM; (C) size distribution of Fe-MOF; (D) size distribution of DOX@Fe-MOF@PEM; (E) zeta
potential of nanoparticles (a: Fe-MOF, b: DOX@Fe-MOF, c: DOX@Fe-MOF@PSS, d: DOX@Fe-MOF@PSS@MV-PAH, e: DOX@Fe-MOF@PEM); (F) N2 isotherm of
pristine Fe-MOF at 77 K; (G) pore size distribution of pristine Fe-MOF derived from N2 isotherm data and (H) the EDX analysis for Fe and O elements of Fe-MOF.
particles at the same dosage of 2.5 mg mLꢂ1 DOX, respectively.
After 24 h of incubation, cells were harvested, washed twice,
3. Results and discussion
3.1. Preparation of MV-PAH
and resuspended with PBS. The Annexin V-FITC apoptosis
detection kit was used for apoptosis, quantified by flow
cytometry.
The preparation of MV-PAH is represented in Scheme 2. First, 1-
methyl-4,40-bipyridinium iodide (1) was prepared by alkylation
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reaction, and the typical 1H NMR spectra is depicted in Fig. 1A. Fe-MOF@PEM were 102 nm and 110 nm (Fig. 2C and D),
As expected, all characteristic peaks could be observed, in respectively. The average size determined by DLS of DOX@Fe-
good agreement with the previous report. Next, complex (1) MOF@PEM was larger than that of Fe-MOF, which was attrib-
was reacted with 3-bromopropionic acid counter-anion, and uted to hydration layers around the particle surface in aqueous
subsequent ion exchange gave MV-COOH (2). Finally, complex solution. Further experiments were performed to measure the
(2) was conjugated to PAH through a condensation reaction zeta potential of the resulting nanoparticles. The polyelectrolyte
of carboxyl and amino groups. As depicted in Fig. 1A, MV-PAH multilayers (PSS and MV-PAH) coating the nanoparticles
exhibited the same chemical shifts at d 8.43–9.07 ppm remarkably changed the zeta potential; the values were 26.9,
as complex 2, which was attributed to aromatic hydrogen 19.8, ꢂ14.2, 16.3 and ꢂ13.5 mV for Fe-MOF, DOX@Fe-MOF,
protons, indicating the successful conjugation of MV-COOH DOX@Fe-MOF@PSS,
DOX@Fe-MOF@PSS@MV-PAH
and
to PAH. Furthermore, as visible in Fig. 1B, the UV absorption DOX@Fe-MOF@PEM, respectively (Fig. 2E). N2 adsorption–
of the MV-grafted polymer had a baseline shift at 257 nm, desorption measurements are depicted in Fig. 2F and G; the
which could be ascribed to the characteristic UV absorbance surface area and pore size were calculated to be 592.2 m2 gꢂ1
peak of 1-(2-carboxyethyl)-1’-methyl-4,4’-bipyridinium dichlor- and 5.4 nm, respectively. The EDX analyses of Fe and O element
ide at this wavelength, suggesting that the successful combi- of Fe-MOF are shown in Fig. 2H. According to the UV-Vis
nation of MV-COOH and PAH was confirmed. To standard curve method at the wavelength of 480 nm, the DLC
determine how much methyl viologen was grafted onto the of DOX in DOX@Fe-MOF@PEM was calculated to be a decent
polymer, a calibration curve of 1-(2-carboxyethyl)-10-methyl-4,40- rate (88.4%), which could be attributed to its large cavity and
bipyridinium dichloride in water was created in Fig. 1C. Using specific surface area.
this technique, it could be determined that on average, six
methyl viologen entities were grafted to one polymer chain.
3.3. Stability and in vitro drug release
3.2. Synthesis and characterization of Fe-MOF nanoparticles
and DOX@Fe-MOF@PEM nanoparticles
The physical stability of DOX@Fe-MOF@PEM was also deter-
mined. After
4 weeks of storage in PBS, the DOX@
In this work, the DOX@Fe-MOF@PEM nanoparticles were Fe-MOF@PEM solution remained clear, without any significant
fabricated via three-step procedure. Initially, Fe-MOF precipitation. In addition, as shown in Fig. 3A, the sizes
a
nanoparticles were formulated according to previous literature remained stable, and the PDI also remained at a favorable
with slight modifications. Then, the prepared Fe-MOF level, which may be due to the stereospecific block of the
nanoparticles were packaged with DOX for anticancer drug PEM. The results reveal the favorable physical stability of
loading. Finally, the formed DOX-loaded Fe-MOF was coated nanoparticles under normal physiological conditions,
with polyelectrolyte multilayers (PSS and MV-PAH) by the layer- indicating they might be used for a long duration of circulation
by-layer technique. TEM images of Fe-MOF and DOX@Fe- time in the bloodstream. The release efficiency of DOX under
MOF@PEM nanoparticles are displayed in Fig. 2A and B. The physiological conditions of PBS (pH 7.4 and 5.0) was fully
Fe-MOF nanoparticles were produced from an aqueous reaction investigated. As depicted in Fig. 3B, DOX@Fe-MOF@PEM
mixture of FeCl3ꢀ6 H2O and H2N–BDC, with needle-shaped released less than 4% of DOX in PBS at pH 7.4, while the
morphology of an average length of 95 nm and width of nanoparticles released nearly 72% of DOX in PBS at pH 5.0 after
42 nm. Compared with Fe-MOF, the size of DOX@ 12 h of incubation. Once the pH was lowered, significant
Fe-MOF@PEM was almost same as the unmodified example, sustained release could be observed, and the release efficiency
but the surfaces have coated polyelectrolyte multilayers of was positively correlated with time, further confirming that
B3 nm. Moreover, dynamic light scattering measurements PEM-encapsulated nano-ions could effectively control drug
for the hydrodynamic sizes of Fe-MOF and DOX@ release.
Fig. 3 (A) Changes in the size and PDI of DOX@Fe-MOF@PEM stored at 4 1C in PBS; (B) in vitro DOX release profiles of DOX@Fe-MOF@PEM in PBS.
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intrinsic red fluorescence of DOX and the blue fluorescent light
of DAPI. As could be observed in Fig. 5, after 4 h of incubation,
the DOX@Fe-MOF group showed a weak red fluorescence,
while the red fluorescence of the DOX@Fe-MOF@PEM
group was stronger. The results could be explained by the
effective encapsulation of drugs by PEM and thus reduced drug
leakage.
3.6. ROS generation
To further evaluate the intracellular ROS generation induced by
various formulations, an indicator, 20,70-dichlorodihydro-
fluorescein diacetate (DCFDA), was employed to detect ROS
generation, and flow cytometry was applied to explore the
changes in the intracellular ROS level of A549 cells. The offset
in Fig. 6A could be related to the amount of produced ROS, and
a significant difference was noticed in Fig. 6B. The quantity of
produced ROS in A549 cells incubated with DOX@
Fig. 4 Absorbance spectra with the addition of Fe-MOF at various
concentrations.
3.4. ꢁOH generation
As a typical chromogenic reagent, TMB usually serves as a Fe-MOF@PEM was 30-fold more than that of the control group
peroxidase to reduce H2O2 to H2O. Likewise, various concentra- and 8.29-fold more than that of DOX. The results indicate that
tions of Fe-MOF have been shown to catalyze the oxidation of DOX@Fe-MOF@PEM could obviously elevate the intracellular
TMB by H2O2. Research has established that these iron com- ROS levels.
pounds first catalyze H2O2 to generate ꢁOH, which oxidizes
3.7. In vitro cytotoxicity
TMB to form ox-TMB. As shown in Fig. 4, as the concentration
of Fe-MOF (0, 5, 10, 20, 40, 80 mg mLꢂ1) increased, the UV
absorbance increased. Moreover, the inset shows that the
relative absorbance of ox-TMB is a function of Fe-MOF coex-
istence at 370 and 652 nm.
To assess the antitumor effects of the synthesized nano-
particles, the cytotoxic activity of different formulations was
evaluated by MTT assays on A549 and MCF-7 cell lines. As
presented in Fig. 7, the Fe-MOF nanoparticles incubated with
cancer cells exhibited high cell viability at various concentra-
tions, which firmly verified its relatively low cytotoxicity against
3.5. Cellular uptake
The internalization of DOX@Fe-MOF and DOX@Fe-MOF@PEM cancer cells. As expected, DOX@Fe-MOF at 40 mg mLꢂ1 concen-
was assessed by CLSM to qualitatively evaluate the cellular tration of DOX displayed a stronger inhibitory effect against
uptake of DOX@Fe-MOF@PEM by A549 cells, utilizing the the cells (83.9% for MCF-7 cells and 83.3% for A549 cells).
Fig. 5 CLSM images of the A549 cells cultured with DOX@Fe-MOF and DOX@Fe-MOF@PEM. Scale bar: 20 mm.
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Fig. 6 (A) Quantification ROS intensity of A549 cells induced by different drug formulations, measured by flow cytometry; (B) the analysis of flow
cytometry data (a: Control, b: DOX, c: Fe-MOF, d: DOX@Fe-MOF, e: DOX@Fe-MOF@PEM). n = 3, mean ꢄ SD; ***p o 0.001.
Fig. 7 Relative cell viability of (A) A549 and (B) MCF-7 cells incubated with various formulations. n = 3, mean ꢄ SD; *p o 0.05; ***p o 0.001.
Fig. 8 Apoptosis of A549 cells induced by different formulations achieved using Annexin V-FITC/PI staining. n = 3, mean ꢄ SD; *** p o 0.001.
Nevertheless, the cell inhibition ratio of DOX@Fe-MOF@PEM for 24 h was determined by flow cytometry. As depicted in Fig. 8,
was 91.6% for MCF-7 cells and 91.4% for A549 cells, which was the apoptosis in Fe-MOF displayed a special low rate (3.34%),
indicative of the role of PEM catalysis in cytotoxicity. In indicating that Fe-MOF is an extraordinarily safe, biocompatible
summary, the results revealed that DOX, MOF and PEM possess carrier. Compared with 22.39% of apoptosis induced by free DOX,
synergism and heightened cytotoxicity.
the cells cultured with DOX@Fe-MOF and DOX@Fe-MOF@PEM
exhibited a notable apoptosis ratio of 39.79% and 62.9%, respec-
tively. The outcomes indicate the role of Fe-mediated cell
3.8. Cell apoptosis
To further explore the effect of nanoparticle-induced apoptosis of apoptosis and clearly reveal that the prepared DOX@Fe-
A549 cells, the percentage of apoptotic cells in samples treated MOF@PEM could significantly induce apoptosis of A549 cells by
with Fe-MOF (the control group), free DOX, DOX@Fe-MOF, and the synergy of DOX, MV, and Fe-MOF, which is consistent with the
DOX@Fe-MOF@PEM at the identical dose of 2.5 mg mLꢂ1 DOX observations from the in vitro cytotoxicity test.
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4. Conclusions
In summary, a cationic polymer MV-PAH was synthesized
rationally, and then a new pH-responsive nanocarrier based
on the biocompatible Fe-MOF was effectively built for carrying
DOX modified with the cationic polymer MV-PAH multilayers
prepared by LBL technique. The PEM decomposed in the
weakly acidic environment of tumor cells, significantly enhan-
cing drug delivery. DOX@Fe-MOF@PEM noticeably inhibited
the cancer cells in the cytotoxicity and apoptosis experiments.
The H2O2 induced by DOX and MV-PAH could catalyze the
formation of highly toxic ꢀOH from iron ions in cells through a
Fenton-like reaction, simultaneously increasing the anticancer
effect of DOX. This original strategy has created a potential way
to provide a synergistic effect of ROS and developed a suitable
chemotherapy for enhanced anti-cancer efficacy.
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Conflicts of interest
There are no conflicts to declare.
Acknowledgements
This work was financially supported by the National Natural
Science Foundation of China (Grant No. 21628101 and
21371031), the International S&T Cooperation Program of
China (No. 2015DFG42240) and the Priority Academic Program
Development (PAPD) of Jiangsu Higher Education Institutions.
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