The synthesis of LA-Fe3O4@PDA-PEG-DOX for photothermal therapy-chemotherapy

Article abstract of DOI:10.1039/c7dt04080f

A facile methodology is presented to construct a multifunctional nanocomposite that integrates photothermal therapy and specific drug release into a single nanostructure. Firstly, magnetic Fe₃O₄@polydopamine core-shell nanoparticles (Fe₃O₄@PDA) were synthesized via a reversed-phase microemulsion approach. By varying the amount of DA, Fe₃O₄@PDA with a particle size of 28-38 nm can be obtained. To further ensure the monodispersity, biocompatibility and specific uptake, PEG and lactobionic acid (LA) were grafted onto Fe₃O₄@PDA (LA-Fe₃O₄@PDA-PEG), whose fast photothermal conversion is derived by the combination of Fe₃O₄ and PDA with high near infrared (NIR) absorption. Then, doxorubicin hydrochloride (DOX) was adopted as the typical anticancer drug, which was loaded onto LA-Fe₃O₄@PDA-PEG via electrostatic and π-π stacking interaction. The release kinetics investigation further demonstrated the acid/heat-triggered DOX release. HepG2 cells (hepatocellular cell line) were used as the target cancer cells, and the fast uptake was due to the nanoparticle size and abundant asialoglycoprotein receptors on HepG2 cells. Besides, an external magnetic field also can improve the uptake, especially when the magnet is placed at the bottom of the cell disk. The enhanced specific cytotoxicity toward HepG2 cells was also ascribed to the synergistic effect of chemo- and photothermal therapy. Based on the novel properties, the LA-Fe₃O₄@PDA-PEG-DOX nanocomposite showed its potential application in hepatocyte therapy.

Full text of DOI:10.1039/c7dt04080f

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The Synthesis of LA-Fe3O4@PDA-PEG-DOX for Photothermal-Chemotherapy
Therapy
Yuhua Chen, Huiming Lin,* Feng Zhang, Qian Wang, Ruihan Tong, Na An, and
Fengyu Qu*
Key Laboratory of Photochemical Biomaterials and Energy Storage Materials,
Heilongjiang Province , College of Chemistry and Chemical Engineering, Harbin
Normal University, Harbin, 150025, P. R. China
Tel (Fax): +86 451 88060653
E-mail: qufengyu@hrbnu.edu.cn and linhuiming@hrbnu.edu.cn
1

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ABSTRACT
A facile methodology is presented to construct a multifunctional nanocomposite,
which integrates photothermal therapy and specific drug release into a single
nanostructure. Firstly, the magnetic Fe3O4@polydopamine coreꢀshell nanoparticles
(Fe3O4@PDA) were synthesized via reversedꢀphase microemulsion approach. By
varying the amount of DA, Fe3O4@PDA with particle size of 28ꢀ38 nm can be
obtained. To further make sure the monodispersity, biocompatibility and specific
uptake, PEG and lactobionic acid (LA) were modified on to Fe3O4@PDA
(LAꢀFe3O4@PDAꢀPEG), whose fast photothermal conversion is derived the
combination of Fe3O4 and PDA with high near infrared (NIR) absorption. And then,
doxorubicin hydrochloride (DOX) was adopted as the typical anticancer drug, which
was loaded on to LAꢀFe3O4@PDAꢀPEG via electrostatic and π–π stacking interaction.
The release kinetics investigation further demonstrated the acid/heatꢀtriggered DOX
release. HepG2 cells (hepatocellular cell line) were used as the target cancer cells, and
the fast uptake was owing to the nanoparticle size and abundant asialoglycoprotein
receptor on HepG2 cells. Besides, the external magnetic field also can improve the
uptake, especially, when the magnet was placed at the bottom of the cell disk. The
enhanced specific cytotoxicity toward HepG2 cells was also ascribed to the
synergistic effect of chemoꢀ and photothermal therapy. Based on the novel properties,
LAꢀFe3O4@PDAꢀPEGꢀDOX nanocomposite showed the potential application on
hepatocyte therapy.
Keywords: Fe3O4@PDA; Nanocomposite; NIR; LA target; Chemotherapy;
Photothermaltherapy
2

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1. INTRODUCTION
Recently, nearꢀinfrared (NIR) laserꢀmediated photothermal therapy (PTT) has
been considered as a promising approach for anticancer due to its deep tissue
penetration, improved target selectivity and therapeutic efficacy as well as minimal
invasiveness and side effects. Up to now, numerous photothermal agents, such as gold,
carbon, copper sulfide nanostructures have been adopted to converse NIR into thermal
lethality for the treatment of tumors. Based on the previous reports, only PTT is not
effective enough, therefore, synergistic therapy which integrates PTT with some other
therapeutic approaches into one nanoplatform has shown great potential for antitumor
treatment. Zhang et al. facilitated carbon nanoparticles/doxorubicin@SiO2
nanocomposites as an efficient nanoplatform for combined cancer photothermal and
chemoꢀtherapy.1 Wang et al. developed multifunctional gold nanorod@silicaꢀcarbon
dots (GNR@SiO2ꢀCDs) as a phototheranostic agent. In GNR@SiO2ꢀCDs, the GNRs
act as photothermal therapy (PTT) agents, and the CDs serve as photodynamic
therapy (PDT) agents, which can be achieved to guide PDT/PTT treatments.2 Zhao et
al. developed AuNRꢀSiꢀZnPc nanoplatform for PDT and PTT dualꢀmodal therapy,
where AuNR served as photothermal therapy (PTT) agents, ZnPc act as photodynamic
therapy (PDT) agents and the antitumor efficiency was significantly enhanced via the
combination therapy.3
With excellent biocompatibility, easy surface modification, and strong NIR
absorption polydopamine (PDA) has recently received substantial interests on
potential application of tissue engineering, biosensing, bioimaging, drug and gene
delivery, and photothermal treatment.4ꢀ7 Lu et al. have recently demonstrated that
PDA nanospheres can be prepared via selfꢀpolymerization of dopamine，showing
desirable biocompatibility and the greater photothermal conversion efficiency than
gold nanorods.8 Polydopamine (PDA) was developed as a promising carrier for
loading of both radionuclides and an anticancer drug to realize
nuclearꢀimagingꢀguided combined radioisotope therapy (RIT) and chemotherapy of
cancer in one system.9 Park and colleagues have prepared polycaprolactone (PCL)
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nanofibers modified by gelatin and PDA thin films that were used as the scaffolds for
human endothelial cell growth, revealing enhanced adhesion and viability, positive
expression of endothelial cell markers and increased stress fiber formation.10 Lin et al.
developed a facile approach for modifying ICG based on selfꢀpolymerization of
dopamine coated on the UCNP nanoparticles surface to achieve PTT/PDT dualꢀmodal
therapy.11
Fe3O4 NPs possess outstanding properties, including excellent magnetism,
biocompatibility, highly targeting ability and photothermal conversion efficiency, and
also have widely used in biomedicine. However, bare Fe3O4 NPs are prone to
aggregation because of anisotropic dipolar attraction and rapid biodegradation when
they are exposed directly to biological systems.12ꢀ25 Up to new, Much effort also has
been devoted to the fabrication of multifunctional nanocomposites, such as graphene
oxideꢀFe3O4,26 Fe3O4@Au,27 and Fe3O4@Cu2xS.28 And the magnetic nanoparticles
coated with lipid, polymers, and proteins were also designed as magnetically guided
nanocomposites.29ꢀ32
Herein, a facile methodology is presented to construct multifunctional
nanocomposite, which integrates photothermal therapy and tumorꢀtargeted drug
delivery into one nanostructure. In this work, Fe3O4 NPs were synthesized as core
coated with polydopamine (PDA) shell to construct Fe3O4@PDA NPs with uniform
particle size (28ꢀ38 nm) via a reversedꢀphase microemulsion method (Scheme 1A).
Then, amineꢀterminated PEG was applied in functionalizing Fe3O4@PDA
nanoparticles via the Michael addition and Schiff base reaction (Scheme S1) to
promote their monodispersity and uptake by cell. And then, doxorubicin
hydrochloride (DOX) would be loaded onto LAꢀFe3O4@PDAꢀPEG via hydrophobic
and π–π stacking interaction. Under 808 nm irradiation, the high NIR absorption of
PDA and Fe3O4 further induce the elevated photothermal conversion efficiency of
Fe3O4@PDA. Moreover, the heat also reduces the interaction between PDA and DOX,
making DOX release. In order to further improve the targeting, lactobionic acid (LA)
was grafted outside. Hepatocellular cell line (HepG2 cells) was adopted as typical
cancer cell and fast uptake of LAꢀFe3O4@PDA3ꢀPEGꢀDOX is derived from the
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asialoglycoprotein receptor, small particle size, and external magnetic field. The
synergistic effect of chemotherapy and PTT associated with fast uptake further make
sure the enhanced specific cytotoxicity to hepatocyte cells.
2. EXPERIMERENTAL SECTION
2.1 Materials. Unless specified, all of the chemicals utilized were analytical grade
and utilized without further purification. Iron chloride (FeCl3·H2O), sodium oleate,
trioctylamine, 1ꢀoctadecene, COꢀ520, dopamine hydrochloride, Methoxypolyethylene
glycol amine (mNH2ꢀPEG), Nꢀ(3ꢀdimethylaminopropyl)ꢀNꢀethylcarbodiimide
hydrochloride (EDC), Nꢀhydroxysuccinimide (NHS), lactobionic acid (LA) and
doxorubicin hydrochloride (DOX) were all purchased from Aladdin of China.
2.2 Instruments. Samples were characterized by transmission electron microscopy
(TEM Hitachi Hꢀ8100) at an accelerating voltage of 20 kV. Xꢀray diffraction (XRD)
is characterized using a Rigaku Ultima IV diffractometer with Cu Ka radiation (40kV,
20 mA). The fluorescence spectra were surveyed by HORIBA FLꢀ3. A Fourier
transform infrared (FTꢀIR) spectroscopy spectrometer (JASCOFT/IRꢀ420) was used
to record the infrared spectra of these materials. Zeta potential was carried out on a
ZetaPALS Analyzer. Ultravioletꢀvisible (UVꢀVis) spectra were taken on (SHIMADZU
UV2550 spectrophotometer). The isotherms of N2 adsorption/desorption were
measured at the temperature of liquid nitrogen using a Micromeritics ASAP 2010M
system. The pore size distributions were calculated from the adsorption branches of
the N2 adsorption isotherms using the BarrettꢀJoynerꢀHalenda model.
2.3 Synthesis of iron-oleate complex
The metalꢀoleate complex was prepared by reacting metal chlorides and sodium
oleate. In a typical synthesis of ironꢀoleate complex, 10.8 g of iron chloride and 36.5 g
of sodium oleate was dissolved in a mixture solvent composed of 80 mL ethanol, 60
mL distilled water and 140 mL hexane. The resulting solution was heated to 70 °C
and kept at that temperature for four hours. When the reaction was completed, the
upper organic layer containing the ironꢀoleate complex was washed three times with
30 mL distilled water in a separatory funnel. After washing, hexane was evaporated
off, resulting in ironꢀoleate complex in a waxy solid form.
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2.4 Synthesis of iron oxide nanocrystals (Fe3O4)
The following is a typical synthetic procedure for monodisperse iron oxide
(magnetite) nanocrystals. 36 g of the ironꢀoleate complex synthesized as described
above and 5.7 g of oleic acid were dissolved in 200 g of trioctylamine at room
temperature. The reaction mixture was heated to 360 °C with a constant heating rate
of 3.3 °C min–1, and then kept at that temperature for 30 min. When the reaction
temperature reached 360 °C, a severe reaction occurred and the initial transparent
solution became turbid and brownish black. The resulting solution containing the
nanocrystals was then cooled to room temperature, and 500 mL of ethanol was added
to the solution to precipitate the nanocrystals. The nanocrystals were separated by
centrifugation.
2.5 PDA-coated iron oxide nanocrystals (Fe3O4@PDAs)
10 mL of the asꢀobtained Fe3O4 cyclohexane solution was mixed with 0.65 ꢁL of
Igepal COꢀ520 before the addition of ammonium hydroxide (100 ꢁL 25 wt%),
followed by adding 30, 45, 60, 75 ꢁL of dopamine hydrochloride aqueous solution
(25 wt%). The asꢀobtained Fe3O4@PDAs nanoparticles were collected by
centrifugation and washed with water for several times until no color in the
supernatant.
2.6 Synthesis of Fe3O4@PDAs-PEG
Fe3O4@PDAsꢀPEG (Scheme S1) were prepared by simply mixing Fe3O4@PDAs
with mPEGꢀNH2 at a mass ratio of 1:2 in alkaline buffer solution (pH = 9) under
magnetic stirring for 24 h. The product was purified by centrifugation and washed
with DI water for three times.
2.7 LA peptide attachment. 1 mmol of Fe3O4@PDAsꢀPEG and LA peptide were
dissolved in 2 mL of DMSO followed by the addition of NHS and EDC as catalyst,
and then the mixture was stirred at 25°C for 12 h to obtain LAꢀFe3O4@PDAsꢀPEG.
The excess NHS, EDC and LA were removed by repeatedly washing with ethanol.
2.8 DOX Loading and Release
DOX loading onto Fe3O4@PDAs, Fe3O4@PDAsꢀPEG, LAꢀFe3O4@PDAsꢀPEG
were conducted by mixing
4
mL of Fe3O4@PDAs, Fe3O4@PDAsꢀPEG,
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LAꢀFe3O4@PDAsꢀPEG solution (250 mg mLꢀ1) with different amounts of DOX (0.5,
1, 2 mg) in phosphate buffer (20 × 10−3 M ) at pH 7.4. The mixture was stirred for 6 h
in dark. Unbound excess DOX was then removed by centrifugation and washed with
DI water several times. The obtained DOXꢀloaded nanoparticles were dried for next
experiments. The loading amount of DOX was measured by UVꢀVis spectroscopy at
480 nm. The loading efficiency (LE wt%) of DOX can be calculated by the below
formula. All the data are derived from three parallel tests.
m(original DOX) — m residual DOX
(
Loading Efficiency LE% =
) ×100%
(
)
m(original DOX)
For DOX releasing measurement, Fe3O4@PDAsꢀDOX solutions were dialyzed in
PBS at pH 5.0 and 7.4 in dark and kept in a 37 °C water bath. At different time points,
DOX released from Fe3O4@PDAsꢀDOX was collected and measured by UV–Vis
spectra.
2.9 Measurement of Photothermal Performance.
To investigate the photothermal effect of Fe3O4, PDA, Fe3O4@PDAs, 1 mL of
aqueous suspension solutions (125 ug mLꢀ1) were irradiated under an NIR laser (808
nm, 2 W cmꢀ2) for 10 min, respectively. A digital thermometer was used to measure
the temperature during irradiation.
2.10 Cell culture. HepG2 cells (hepatocellucar cell line) were grown in a
monolayer in Dulbecco’s Modified Eagle’s Medium (DMEM, Gibco) supplemented
with 10% (v/v) fetal bovine serum (FBS, Tianhang Bioreagent Co., Zhejiang) and
penicillin/streptomycin (100 U mLꢀ1 and 100 mg mLꢀ1, respectively, Gibco) in a
humidified 5% CO2 atmosphere at 37℃.
2.11 Cell cytotoxicities assay. For the MTT assay, HepG2 cells were seeded into
96ꢀwell plates at a density of 1 × 104 per well in 100 uL of media and grown overnight.
Then, different NPs were added to the medium without/with magnetic field in
different directions (Scheme S2), the cells were incubated for 24 h. And then, the
culture medium with nanoparticles was removed and the cells were resupplied with
fresh culture medium. The cells were irradiated with NIR irradiation (1 W cmꢀ2, 30
min). The cells were further incubated for another 4 h. Afterwards, cells were
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incubated in media containing 0.5 mg mLꢀ1 of MTT for 4 h. The medium was then
replaced with 150 mL of dimethyl sulfoxide (DMSO) per well and the absorbance
was monitored using a microplate reader (BioꢀTekELx800) at the wavelength of 490
nm. The cytotoxicity was expressed as the percentage of cell viability compared to
untreated cells.
2.12 Fluorescence imaging. To analyze the ability of cellular internalization in
different conditions, HepG2 cells were cultured in a 6ꢀwell with the incubation
medium (DMEM) for 24 h, and then treated with/without LA modified
LAꢀFe3O4@PDA3ꢀPEGꢀDOX
under
NIR
irradition
and
LAꢀFe3O4@PDA3ꢀPEGꢀDOX with magnetic field in different directions (Scheme S2)
at concentration of 125 ꢁg mLꢀ1. After the cells were respectively incubation for 6 h,
the medium was removed and the cells were washed twice with PBS, then
glutaraldehyde solution (2.5 %, 1 mL) was added into culture dish for 10 min, the
cells were washed twice with PBS, then the DAPI were added to incubate for 10 min
and the cells were washed twice with PBS. The fluorescence imaging experiments
were carried out on the Leica DFC450 C Microsystems Ltd. using a 10× objective.
2.13 Photothermal therapy. HepG2 cells were cultured in a 6ꢀwell with the
incubation medium (DMEM) for 24 h, and then treated with LAꢀFe3O4@PDA3ꢀPEG
for 6 h. Followly, the calcine AM (500 ꢁL 2 ꢁmol Lꢀ1) was added to incubate for 30
min and propidium iodide (PI) (2 ꢁL 4 ꢁmol Lꢀ1) was added to incubate for 10 min
after irradition with 808 nm laser for 0, 10, 30 min.
3 RESULTS AND DISCUSSION
3.1 Fabrication and characterization of the materials
The morphologies and structures of Fe3O4 and Fe3O4@PDAs were investigated
through TEM analysis. As displayed in Figure 1A, Fe3O4 core shows the dispersed
and uniform spherical morphologies with 22 nm in size. The highꢀresolution (HR)
TEM image (Figure 1A inset) confirms the high crystallinity of the particles with a
dꢀspacing value of 0.25 nm, which is close to the d311 value of Fe3O4. And the large
angle XRD pattern of Fe3O4 exhibits diffraction peaks at 18.3, 30.1, 35.4, and 62.6° is
ascribed to (111), (220), (311), and (440) diffractions Fe3O4 (JCPDS 65ꢀ3107) (Figure
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S1) that is in good agreement with Figure 1A inset. After the coating of PDA, the
coreꢀshell structure of Fe3O4@PDAs can be found from Figure 1BꢀD, remarkably.
With increasing the amount of DA, the thickness of PDA shell aggrandizes to induce
the increased particle size of Fe3O4@PDA1 (28 nm), Fe3O4@PDA2 (32 nm), and
Fe3O4@PDA3 (38 nm) nanoparticles. However, go on increasing DA, Fe3O4@PDA4
nanoparticles show the nonuniform morphologies (Figure 1E). Here, reversedꢀphase
microemulsion method was adopted to prepare the monodisperse Fe3O4@PDA
nanoparticles, but too much DA (aqueous phase) disturbs the microemulsion to make
the nonuniform structure at last. From the EDS graph as shown in Figure 1F, the
emerging N signal and the improved C signal of Fe3O4@PDA3 further demonstrates
the successful coating of PDA.
FTꢀIR spectrometry was used to confirm the drug loading and modification.
As shown in Figure 2A, the strong absorption at 575 cmꢀ1 is related to the typical
vibration of FeꢀO for all samples. After the coating of PDA shell, the enhanced
absorption band at 3441 cmꢀ1 is ascribed to the hydroxyl and amine groups from PDA
(From 2Abꢀc). In addition, the emerging peaks at 1500, 1600, 1250 cmꢀ1 are
corresponding to the stretching vibration C=C and CꢀC from aromatic rings in PDA as
shown in Figure 2Ab. From 2Ac, the improved peaks at 3064 is due to the stretching
vibration of =CꢀH in the additional DOX. And the absorption peak of v(C＝O) at
1680 cmꢀ1 and the marked split peak of v(C＝C) at 1600 and 1580 cmꢀ1, further
confirming the loading of DOX. Besides, the UVꢀVis spectra were also recorded to
further survey the organic modification and drug loading process. As shown in Figure
2B, in comparison to Fe3O4, the enhanced NIR absorption of Fe3O4@PDA is due to
the coating of PDA, which is a typical NIR photosensitizer. Moreover, the absorption
at 480 nm further demonstrates the loading of DOX. In addition, the zeta potentials of
the NPs were determined to further monitor the coating process (Figure 2C). After the
successful coating of PDA shell on the surface of Fe3O4, the mean zeta potential value
decreases to ꢀ0.93±0.41, ꢀ1.02±0.96, and ꢀ5.67±0.52 mV for Fe3O4@PDA1,
Fe3O4@PDA2, and Fe3O4@PDA3, respectively, due to the presence of a large number
of the surface –OH on Fe3O4@PDAs. Further attracting with DOX, the surface
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potential of Fe3O4@PDA3ꢀDOX rises to 6.21±1.34 mV owning to the positive
character of DOX molecules. Magnetization characterization of these samples was
carried out at 300 K (Figure 2D). All the samples possessed superparamagnetism, and
the asꢀsynthesized Fe3O4 nanoparticles show the high saturation magnetization of
60.72 emu gꢀ1. After the assembly process, the saturation magnetization drops to 45.56
(Fe3O4@PDA1), 36.82 (Fe3O4@PDA2), 27.57 (Fe3O4@PDA3) emu gꢀ1 because of the
nonꢀmagnetic component of PDA. With high saturation magnetization, Fe3O4
nanoparticles easily cause the particle aggregation, and the improved stability of
Fe3O4@PDA3 and Fe3O4@PDA3ꢀPEG is good for the further application (Figure S2).
In view of the good superparamagnetism, the magnetic targeting and separation can
be easily realized (the inset of Figure 2D).
To study the photothermal effect of these samples, NIR laser (808 nm, 2 W cmꢀ2)
was introduced. From 3A, all the samples exhibit the increased temperature
depending on irradiation time. The bare Fe3O4 induces temperature up to 36.2
within irradiation for 10 min. After the coating of PDA layer, the temperature of
Fe3O4@PDAs solution is higher than that of bare Fe3O4, and Fe3O4@PDA3 reveals
the fast temperature rise up to 44.3 as the thickest PDA layer. In comparison, the
temperature of pure water was increased by only 2.6
under the same exposure
condition. Furthermore, Fe3O4@PDA3 also possesses considerable photothermal
stability as presented in Figure 3B. The photothermal conversion does not attenuate
obviously after 5 cycles of irradiation, owing to the stable nanostructure of
Fe3O4@PDA3 as shown in Figure 3C. This photothermal effect implied that the
Fe3O4@PDA would be effective photothermal agents for cancer therapy.
3.2 Drug release profiles.
The anticancer drug DOX was used as a model drug to investigate the controlled
release performances of Fe3O4@PDA3. As revealed in Figure 2C, Fe3O4@PDA3
possesses the negative surface charge and the electrostatic interaction between PDA
shell and DOX induces the high loading efficiency (36.91±1.31 %) for DOX. Besides,
the strong π–π stacking also plays a role in the high loading amount. In addition, the
cumulative DOX release profiles from Fe3O4@PDA3 in pH 5.0 and 7.4 PBS are
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shown in Figure 4. As can be seen in Figure 4A, Fe3O4@PDA3 reveals the smart
pHꢀsensitive drug release. In pH 5.0 PBS, a persistent and longꢀterm release behavior
can be surveyed. After 48 h, the release can reach 30.31±1.72 % in pH 5.0, while that
is obviously inhibited at pH 7.4 (5.60±0.63 %). The lower pH condition makes the
surface zetaꢀpotential of PDA more positive and the protonation of the amino group in
the DOX molecule, which weaken the electrostatic interaction between PDA and
DOX molecules, facilitating drug release under acidic pH. The specific release is
significant for the practical application because the pH difference between the normal
and cancer cells. Moreover, when 808 nm NIR laser (2 W cm−2, 10 min for each pulse)
was introduced, a burst release of DOX was observed from Fe3O4@PDA3ꢀDOX at
pH 5.0 (Figure 4B), ascribing to the decreased π–π stacking under hyperthermia
triggered by NIR (Figure S4). After 48 h (with total illumination time to 50 min), the
release is enhanced to 54.89±1.83 %. The burst release depends on the excitation
source, indicating that the release process and amount could be fineꢀtuned by remote
control of the NIR irradiation. Based on the Figure 4, the acid and NIRꢀtriggered
DOX release makes Fe3O4@PDA3ꢀDOX as potential candidate for onꢀdemand drug
release.
3.3 Cellular uptake and cytotoxicity
To observe the cell uptake and in vitro drug release, HepG2 cells were used as
the typical cancer cells. LAꢀFe3O4@PDA3ꢀPEGꢀDOX was incubated with the HepG2
cells for 1, 3, and 6 h under irradiation (1 W cmꢀ1 30 min) (Figure 5) and then the cells
were examined by fluorescence microscopy. As shown in Figure 5,
LAꢀFe3O4@PDA3ꢀPEGꢀDOX nanoparticles can be clearly and largely found in the
cytoplasm of HepG2 cells as demonstrated by the DOX fluorescence lighting up,
indicating that the nanocomposites have been internalized by HepG2 cells through
endocytosis or macropinocytosis (Figure S5). Moreover, the intracellular red
fluorescence also increases with the incubated time. After 6 h, more and more DOX
molecules gather into cell nuclei owing to the acid/NIRꢀenhanced DOX release in
cancer cells that is helpful to improve the drug efficacy because DOX is a cancer drug
acting on the cell nucleus. Besides, the slightly stronger red fluorescence of
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LAꢀFe3O4@PDA3ꢀPEGꢀDOX in compared with that of Fe3O4@PDA3ꢀPEGꢀDOX is
derived from the excess expression of LA receptor on HepG2 cells to make sure the
specific curative effect. And the fast uptake by HepG2 cells is ascribed to the small
nanoparticle size (below 50 nm), the photothermal effect (with NIR laser irradiation)
and the modification of targeting LA.
To further verify the PTT effect, fluorescence microscopy was used to visually
present the viability of HepG2 cells upon incubation with the LAꢀFe3O4@PDA3ꢀPEG
nanocomposites under NIR irradiation. The living cells can be stained with calcein
AM and show green fluorescence, and the dead cells can be stained with PI and show
red fluorescence. As present in Figure 6, the green fluorescence decreases and red
fluorescence increases when the cells were incubated with LAꢀFe3O4@PDA3ꢀPEG
nanocomposites upon exposure to 808 nm laser at a power density of 1.0 W cmꢀ2 for
0ꢀ30 min, indicating a good photothermal cellꢀkilling effect triggered by NIR.
On account of the novel magnetic property, the intracellular uptake efficiency of
LAꢀFe3O4@PDA3ꢀPEGꢀDOX under a external magnetic field was also explored.
After it was incubated with HepG2 cells for 3 h under external magnetic field at
different directions (Scheme S2), the cells were examined by fluorescence microscopy.
From Figure 7, the stronger and more red fluorescence appears in HepG2 cells with
the help of external magnetic field at bottom, suggesting the higher uptaker under
magnetic field (bottom) than the other directions. Those also are further confirmed by
the FCM as shown in Figure S6.
The cell viabilities of HepG2 cells incubated with different samples and
conditions were determined by the MTT assay. As shown in Figure 8A, Fe3O4, PDA,
LAꢀFe3O4@PDA3ꢀPEG shows the negligible cytotoxicity toward HepG2 cells. Even
when the concentration increases to 125 ug mLꢀ1, the cell viability also can remain
above 96.89±2.05%, revealing that the nanocomposite can be used as nanocarrier in
biomedical application. The decreased cell viability of LAꢀFe3O4@PDA3ꢀPEGꢀDOX
(70.26±1.32%) is due to intracellular DOX release under dark condition. As shown in
Figure 8B, upon NIR irradiation, the reduced cell viability of HepG2 cells to
80.21±1.32 % (Fe3O4), 53.03±0.89 % (PDA), 64.58±2.01% (LAꢀFe3O4@PDA3ꢀPEG)
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is ascribed to the 808 nmꢀexcited PTT to induce cell apoptosis. And the further
decreased cell viability to 24.60±1.02 % of LAꢀFe3O4@PDA3ꢀPEGꢀDOX suggests
that the synergistic effect combining chemoꢀ and photothermal therapy exhibits the
enhanced cytotoxicity to HepG2 cells.
As shown in Figure 8C, we investigate the effects of the orientation of magnetic
field. With the help of external magnetic field, the cell viability goes on dropping
from 24.60±1.02 % (in absence of magnetic field) to 16.21±1.52 % (bottom),
22.16±1.74 % (top), and 20.08±1.68 % (side). That is ascribed to the external
magnetic field also supply energy to LAꢀFe3O4@PDAꢀPEGꢀDOX to improve the
endocytosisꢀlike uptake. And when the magnet was place on the bottom of the dish,
the enhanced uptake (as present in Figure 7) made the highest cytotoxicity of all.
In addition, HeLa cell (cervical cancer cell line) was also used as the contrast. As
shown in Figure 8D, there is not obvious distinguish between
LAꢀFe3O4@PDAꢀPEGꢀDOX (52.32±1.93 %) and Fe3O4@PDAꢀPEGꢀDOX
(55.11±2.04 %) incubated with HeLa cells. However, the remarkably decreased
HepG2 cell viability of LAꢀFe3O4@PDAꢀPEGꢀDOX (24.60±1.02 %) in comparison
with Fe3O4@PDAꢀPEGꢀDOX (44.93±1.04 %) is ascribed to the specific recognition
ability of LA to abundant asialoglycoprotein receptor on HepG2 cells. The
controllable and specific cytotoxicity induces the potential application of
LAꢀFe3O4@PDA3ꢀPEGꢀDOX on hepatocyte therapy.
4. CONCLUSION
In conclusion, we have successfully constructed a multifunctional nanocomposite
based on the magnetic Fe3O4 nanoparticle as core and PDA coating as shell
(Fe3O4@PDA). All samples reveal uniform and monodisperse sphere structure with
particle size about 20ꢀ38 nm by adjusting the amount of PDA. Fe3O4@PDA revealed
the fast temperature rise to 44.3 oC under 808 nm irradiation owing to the cooperation
of Fe3O4 and PDA. To further insure the biocompatibility and uptake, PEG and LA
were modified outside. Furthermore, LAꢀFe3O4@PDAꢀPEG is employed as both high
photothermal conversion agent and aromatic anticancer drug carrier. DOX was used
as a model drug, which was loaded onto LAꢀFe3O4@PDAꢀPEG via electrostatic and
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π–π stacking interaction that induced the pH and heat sensitive drug release. In view
of novel magnetic property, LAꢀFe3O4@PDAꢀPEGꢀDOX also exhibits the external
magnetic field enhanced cell uptake. The chemoꢀphotothermal synergistic effect also
caused the improved cytotoxicity. Moreover, the higher specific cytotoxicity to
HepG2 cells than other cancer cells, such as HeLa cells, is derived from the abundant
asialoglycoprotein receptors on HepG2 cells. Based on the above investigation,
LAꢀFe3O4@PDAꢀPEGꢀDOX nanocompositie would possess the potential application
on hepatocyte therapy.
Acknowledgments
Financial support for this study was provided by the National Natural Science
Foundation of China (21471041, 21571045), and College Youth Innovation Talents
Training Program of Heilongjang (UNPYSCTꢀ2015053).
Appendix A. Supplementary material
Supplementary data associated with this article can be found, in the online version
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A
B
Scheme 1. Illustration of the preparation and controlled release /photothermal process
of LAꢀFe3O4@PDAꢀPEGꢀDOX.
C
B
A
50 nm
50 nm
50 nm
D
E
F
50 nm
50 nm
Figure 1. TEM images of (A, inset) Fe3O4, (B) Fe3O4@PDA1, (C) Fe3O4@PDA2, (D)
Fe3O4@PDA3, (E) Fe3O4@PDA4, (F) EDS graph of Fe3O4, Fe3O4@PDA3
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B
A
D
C
Figure 2. FTꢀIR spectra (A) of (a) Fe3O4, (b) Fe3O4@PDA, (c) Fe3O4@PDAꢀDOX.
(B) The UVꢀVis spectra of DOX, Fe3O4, Fe3O4@PDA, Fe3O4@PDAꢀDOX. (C) The
zeta potentials of Fe3O4 and Fe3O4@PDAs. (D) Magnetization characterization of
these samples.
A
B
C
Figure 3. (A) The photothermal cure of diferent samples under irradiation (808 nm, 2
W cmꢀ2) for 10 min. (B) The photothermal stability of Fe3O4@PDA3. (C) The
UVꢀVis adsorption curve of Fe3O4@PDA3 before/after irradiated about 118 min.
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B
A
Figure 4. (A) DOX release from Fe3O4@PDA3 in pH 7.4 and 5.0 in dark. (B) DOX
release from Fe3O4@PDA3 in pH 5.0 and pH 7.4 with irradiation (2W cmꢀ2 10 min for
each pulse).
Figure 5. Fluorescence images of HepG2 cells after incubation for 1, 3, 6 h with
LAꢀFe3O4@PDA3ꢀPEGꢀDOX and Fe3O4@PDA3ꢀPEGꢀDOX under 30 min
irradiation (808 nm 1 W cmꢀ2). For each panel, the images from left to right show
DAPI labeled cell nucleus (blue), DOX fluorescence (red), and then a merged image.
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Figure 6. Fluorescence images of HepG2 cells after incubation for 6 h with
LAꢀFe3O4@PDA3ꢀPEG under different irradiation time (0, 10, 30 min). For each
panel, the images from top to bottom show the pi labeled dead cells (red) and then a
merged image (coꢀstained by Calcine AM and propidium iodide (pi)).
Figure 7. Fluorescence images of HepG2 cells after incubation for 3 h with
LAꢀFe3O4@PDA3ꢀPEGꢀDOX+NIR (808 nm 1 W cmꢀ2 30 min) under different
direction of magnetic field (top, side, bottom). For each panel, the images from left to
right show the bright, DAPI labeled cell nucleus (blue), DOX fluorescence (red), and
then a merged image.
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A
B
D
C
Figure 8. MTT cell viability assay of HepG2 cells after treated by (A) Fe3O4, PDA,
LAꢀFe3O4@PDA3ꢀPEG, LAꢀFe3O4@PDA3ꢀPEG (B) with irradiation (C)
LAꢀFe3O4@PDA3ꢀPEGꢀDOX, LAꢀFe3O4@PDA3ꢀPEGꢀDOX under different
direction of magnetic field with irradiation (D) HepG2 cells and HeLa cells were
treated by LAꢀFe3O4@PDA2ꢀPEGꢀDOX, Fe3O4@PDA2ꢀPEGꢀDOX. Irradiation
experiment: 808 nm irradiation 1W cmꢀ2 for 30 min.
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TOC
The imrpved cell uptake and the synergistic effect of chemo/photothermal therapy
make sure the enhanced specific cytotoxicity toward HepG2 cells.