Aminated β-cyclodextrin-grafted Fe3O4-loaded gambogic acid magnetic nanoparticles: Preparation, characterization, and biological evaluation

Article abstract of DOI:10.1039/c9ra04955j

Based on aminated β-cyclodextrin (6-NH₂-β-CD)-grafted Fe₃O₄ and gambogic acid (GA) clathrate complexes, a nanoparticle delivery system was developed with the aim to achieve low irritation, strong targeting, and high bioavailability of a gambogic acid magnetic nanopreparation. 6-NH₂-β-CD grafted onto Fe₃O₄ MNPs was demonstrated by high-resolution transmission electron microscopy, Fourier transform infrared spectroscopy, X-ray diffraction, zeta potential, and magnetic measurements. The average particle size of the Fe₃O₄?NH₂-β-CD MNPs was 147.4 ± 0.28 nm and the PDI was 0.072 ± 0.013. The encapsulation efficiency, drug loading, zeta potential, and magnetic saturation values of the Fe₃O₄?NH₂-β-CD MNPs were 85.71 ± 3.47%, 4.63 ± 0.04%, -29.3 ± 0.42 mV, and 46.68 emu g^-1, respectively. Compared with free GA, the in vitro release profile of GA from Fe₃O₄?NH₂-β-CD MNPs was characterized by two phases: an initial fast release and a delayed-release phase. The Fe₃O₄?NH₂-β-CD MNPs displayed continuously increased cytotoxicity against HL-60 and HepG2 cell lines in 24 h, whereas the carrier Fe₃O₄?NH₂-β-CD MNPs showed almost no cytotoxicity, indicating that the release of GA from the nanoparticles had a sustained profile and Fe₃O₄?NH₂-β-CD MNPs as a tumor tissue-targeted drug delivery system have great potential. Besides, blood vessel irritation tests suggested that the vascular irritation could be reduced by the use of Fe₃O₄?NH₂-β-CD MNPs encapsulation for GA. The t_1/2 and the AUC of the Fe₃O₄?NH₂-β-CD?GA MNPs were found to be higher than those for the GA solution by approximately 2.71-fold and 2.42-fold in a pharmacokinetic study, respectively. The better biocompatibility and the combined properties of specific targeting and complexation ability with hydrophobic drugs make the Fe₃O₄?NH₂-β-CD MNPs an exciting prospect for the targeted delivery of GA.

Full text of DOI:10.1039/c9ra04955j

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PAPER
Aminated b-cyclodextrin-grafted Fe3O4-loaded
gambogic acid magnetic nanoparticles:
preparation, characterization, and biological
evaluation
Cite this: RSC Adv., 2019, 9, 27136
a
a
a
Wei Fang,†a Ya Ji Dai,†ab Ting Wang, Hai Tao Gao, Peng Huang, Juan Yu,a
*
ac
He Ping Huang,a Dian Lei Wang
and Wei Lu Zonga
*
Based on aminated b-cyclodextrin (6-NH2-b-CD)-grafted Fe3O4 and gambogic acid (GA) clathrate
complexes, a nanoparticle delivery system was developed with the aim to achieve low irritation, strong
targeting, and high bioavailability of a gambogic acid magnetic nanopreparation. 6-NH2-b-CD grafted
onto Fe3O4 MNPs was demonstrated by high-resolution transmission electron microscopy, Fourier
transform infrared spectroscopy, X-ray diffraction, zeta potential, and magnetic measurements. The
average particle size of the Fe3O4@NH2-b-CD MNPs was 147.4 ꢀ 0.28 nm and the PDI was 0.072 ꢀ
0.013. The encapsulation efficiency, drug loading, zeta potential, and magnetic saturation values of the
Fe3O4@NH2-b-CD MNPs were 85.71 ꢀ 3.47%, 4.63 ꢀ 0.04%, ꢁ29.3 ꢀ 0.42 mV, and 46.68 emu gꢁ1
,
respectively. Compared with free GA, the in vitro release profile of GA from Fe3O4@NH2-b-CD MNPs
was characterized by two phases: an initial fast release and a delayed-release phase. The Fe3O4@NH2-b-
CD MNPs displayed continuously increased cytotoxicity against HL-60 and HepG2 cell lines in 24 h,
whereas the carrier Fe3O4@NH2-b-CD MNPs showed almost no cytotoxicity, indicating that the release
of GA from the nanoparticles had a sustained profile and Fe3O4@NH2-b-CD MNPs as a tumor tissue-
targeted drug delivery system have great potential. Besides, blood vessel irritation tests suggested that
the vascular irritation could be reduced by the use of Fe3O4@NH2-b-CD MNPs encapsulation for GA.
The t1/2 and the AUC of the Fe3O4@NH2-b-CD@GA MNPs were found to be higher than those for the
GA solution by approximately 2.71-fold and 2.42-fold in a pharmacokinetic study, respectively. The
better biocompatibility and the combined properties of specific targeting and complexation ability with
hydrophobic drugs make the Fe3O4@NH2-b-CD MNPs an exciting prospect for the targeted delivery of GA.
Received 1st July 2019
Accepted 30th July 2019
DOI: 10.1039/c9ra04955j
rsc.li/rsc-advances
Magnetic drug-loaded nanoparticles have become one of the
research hotspots in current drug delivery systems due to their
non-invasive and high targeting properties.7 Especially, the
1. Introduction
Gambogic acid (GA, C38H44O8) is the major active ingredient of
gamboge,1 which has various biological activities, such as
antipyretic, analgesic, anti-inammatory, autophagic, and anti-
tumor.2 Previous studies have shown that GA can activate
impaired apoptotic pathways in cancerous cells via the down-
regulation of telomerase to achieve an anticancer effect.3
However, the clinical development and applications of GA are
limited to date due to its poor water solubility and bioavail-
ability.4 The use of chemical structure modication methods
could solve these problems.5,6
immobilization of some molecules onto magnetic nano-
particles has attracted considerable attention, such as macro-
cyclic host molecules.8 The aim of such targeted delivery is to
load a drug onto highly responsive magnetic nanoparticles, and
use the external magnetic eld to move and concentrate the
nanoparticles on the target organ or the target tissue, thereby
increasing the drug concentration and improving the bioavail-
ability of the drug.9
Due to their superparamagnetism and biocompatibility,
Fe3O4 magnetic nanoparticles (Fe3O4 MNPs), as magnetic
materials, represent an important component,10 which exhibit
many potential applications, including bioseparation, protein
adsorption, enzyme immobilization, magnetic resonance
imaging, and drug targeting.11 Fe3O4 MNPs have properties of
good chemical stability, biocompatibility, and dispensability in
various solvents.12 Many materials, such as noble metals,
aThe College of Pharmacy, Anhui University of Chinese Medicine, Hefei, 230012,
Anhui, China. E-mail: great7701@126.com; dlwang@ahtcm.edu.cn; Fax: +86-0551-
68129028; Tel: +86-0551-68129159
bAnhui Second People's Hospital, Hefei, 230041, Anhui, China
cAnhui Province Key Laboratory of Chinese Medicinal Formula, Hefei, 230012, Anhui,
China
† The two authors contributed equally to this work.
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surfactants, and polymers, as functional groups have been cyclodextrin (b-CD, W402826, Shanghai Sinopharm Chemical
introduced onto Fe3O4 MNPs for new chemical modication, Reagent Co., Ltd.); methanol (M120521, Shanghai Aladdin
and applications.13
Biochemical Technology Co., Ltd.); and phosphoric acid
b-Cyclodextrin (b-CD) has primary and secondary hydroxyl (P816342, Shanghai Aladdin Biochemical Technology Co., Ltd).
groups,14 and has been used as a natural supramolecular host All reagents were of analytical grade. Water used was deionized
with a cyclic structure, a hydrophilic outer surface and hydro- water.
phobic cavity.15 In the eld of chemistry, pharmaceuticals,
enzyme mimics, and drug carriers were widespread applica-
tion16 where b-CD has potential, as the cavity of b-CD and
a small molecule drug could form an inclusive complex, which
could enhance the solubility of hydrophobic drugs and reduce
undesirable smell and side effects.17 The cyclodextrins of the
host–guest type complexation and their applications in many
elds have recently received much attention.18
Gambogenic acid (GNA)-loaded folic acid (FA)-armed MNPs
(FA-GNA-MNPs) were prepared by our research team, and we
conrmed that FA and GNA were successfully conjugated on the
Fe3O4 core and exhibited substantial inhibitory effects in HeLa
cancer cells.19 Recently, a drug delivery system with the inclu-
2.2 Cells
HepG2 cells, HL-60 cells (China Center for Type Culture
Collection); fetal bovine serum (FBS) (GIBCO, No. 16000-044);
methyl sulfoxide (US Sigma Corporation, 20170210); dimethyl
sulfoxide (US Sigma Corporation, 20170610); thiazolyl blue
tetrazolium bromide (MTT, American Sigma Company, lot
number MKB06849V); Iscove's modied Dulbecco's medium
(IMDM, US HyClone, AB212851); and phosphate-buffered saline
(PBS, prepared in our laboratory).
2.3 Animals
sion property of cyclodextrin, the bioadhesive property of GA, All protocols and animal care were authorized by the Animal
and the magnetic property of iron oxide was successfully Care and Use Committee of Anhui University of Chinese
designed.18 In this paper, Fe3O4@NH2-b-CD-encapsulated GA Medicine and were in accordance with the Guidelines for the
magnetic nanoparticles (Fe3O4@NH2-b-CD@GA MNPs) were Use of Laboratory Animals. All rats and rabbits were purchased
prepared via a co-precipitation method and characterized by from the Anhui Medical University Experimental Animal Center
high-resolution transmission electron microscopy (HRTEM), (Hefei, China). All rats and rabbits were acclimated in an animal
Fourier transform infrared spectroscopy (FTIR), vibrating breeding room under specic pathogen-free (SPF) conditions
sample magnetometry (VSM), and zeta potential.20 The study (Laboratory License No. SYXK (Wan) 2017-0001). Animal
and evaluation of the MNP system's drug-carrying capacity and Certicate No. 201800222. Laboratory animal production
release performance was carried out by the dialysis method. The license No. SCXK (Wan) 2018-0012.
hepatocellular carcinoma cell line HepG2 and leukemia HL-60
cell line were used as research objects. The MTT assay was
used to explore the anti-tumor effect of the nano drug-loading
system on solid tumor cells and non-solid tumor cells. The
pathological tissue sections of rabbit ear veins were observed by
HE staining to measure the vascular irritancy of the gambogic
acid magnetic Fe3O4@NH2-b-CD nanoparticles. The plasma
concentration–time curve of GA in rat plasma was measured to
evaluate the pharmacokinetic characteristics of the
Fe3O4@NH2-b-CD@GA MNPs. It was demonstrated that the
water solubility and bioavailability of gambogic acid were
improved, and the application scope was expanded. This system
can be a promising vehicle for the administration of hydro-
phobic drugs.
2.4 Synthesis of 6-NH2-b-CD
b-Cyclodextrin (5.0 g) was dissolved in 40 mL deionized water,
and NaOH solution was added dropwise until the solution
claried. Next, p-toluenesulfonyl chloride solution containing
acetonitrile (0.84 g p-TosCl, 3 mL CH3CN) was added and stirred
ꢂ
for 4 h at 10 C. Aer the solution was ltered, the ltrate was
adjusted to pH 6, and a white precipitate was obtained as 6-
OTos-b-CD. 6-OTos-b-CD and NaN3 were then added to 15 mL
ethyl alcohol solution and allowed to undergo a reux reaction
for 15 h. Following distillation, the precipitate was dissolved in
100 mL deionized water and ltered, and the ltrate was added
into 100 mL acetone to give a white precipitate, which was
washed with acetone/water (v/v ¼ 5 : 1) to give 6-N3-b-CD. Next,
6-N3-b-CD and (C6H5)3P were added to 2 mL DMF and stirred for
1 h, then 2 mL NH3$H2O was added and the solution was stirred
2. Materials and methods
2.1 Chemicals and reagents
ꢂ
for 4 h at 20 C. Aer the solution was ltered, the ltrate was
poured into 80 mL acetone and a white precipitate was obtained
Gambogic acid (98%, Sigma-Aldrich, St. Louis, MO, United
States); FeCl3$6H2O (F102739, Shanghai Macklin Biochemical
Co., Ltd); FeCl2$4H2O (RA10220008, BeiJing HengYe Zhon-
gYuan Chemical Co., Ltd); ammonium hydroxide (A801005,
as 6-NH2-b-CD.21
2.5 Synthesis of Fe3O4@NH2-b-CD MNPs
Shanghai Macklin Biochemical Co., Ltd); tosyl chloride FeCl3$6H2O (2.61 g) and FeCl2$4H2O (1.05 g) were dissolved in
(T821340, Shanghai Macklin Biochemical Co., Ltd); NaOH, 100 mL deionized water to produce a solution with the
CH3CN, NaN3, (C6H5)3P, N,N-dimethylformamide (Shanghai concentrations of 0.01 mol Lꢁ1 and 0.005 mol Lꢁ1, respectively.
Aladdin Biochemical Technology Co., Ltd); citric acid (C805022, NH3$H2O (W/W: 25%) and citric acid were added at 80 ꢂC until
Shanghai Macklin Biochemical Co., Ltd); ethanol (E164502, the solution turned dark.22 Fe3O4 MNPs were added into the
Shanghai Aladdin Biochemical Technology Co., Ltd); b- MES solution. Subsequently, EDCI (191 mg) and NHS (115 mg)
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were added and stirred for 1 h. The solution of MES containing
6-NH2-b-CD (1.1 g) was added and allowed to react for 12 h. The
nal product was Fe3O4@NH2-b-CD MNPs.20
2.9 In vitro GA release studies
The in vitro release of GA was carried out in PBS (pH 7.4) con-
taining 1% Tween 80. Tween 80 was used to increase the solu-
bility of GA in the buffer solution. The GA solution and
Fe3O4@NH2-b-CD@GA MNPs were transferred into a pre-
swelled dialysis bag (21 mm, molecular weight cut off 8000–
14400 Da, USA), and subsequently, the dialysis bags were sealed
and put into 100 mL release medium at 37 ꢀ 0.5 ꢂC and stirred
at 100 rpm. At predetermined time intervals, 2 mL dissolution
medium was withdrawn and replenished with the same amount
of fresh medium. The amount of GA released was analyzed by
HPLC.
2.6 Preparation of Fe3O4@NH2-b-CD@GA MNPs
The Fe3O4@NH2-b-CD MNPs were prepared using the following
procedure: Fe3O4@NH2-b-CD (40 mg) was dissolved in 15 mL
PBS at pH 7.4. GA (20 mg) was dissolved in 10 mL methanol.
Subsequently, the two solutions were mixed and stirred at room
temperature for 48 h. Following centrifugation, the precipitate
was lyophilized using a vacuum freeze dryer for 24 h to obtain
the Fe3O4@NH2-b-CD@GA MNPs.
2.10 Cell viability and proliferation
The in vitro cytotoxicity of Fe3O4@NH2-b-CD@GA MNPs against
APL HL-60 and HepG2 cells was evaluated using the MTT assay
in the following three experimental groups: GA, Fe3O4@NH2-b-
CD, and Fe3O4@NH2-b-CD@GA MNPs group.24,25 The cytotox-
icity of HL-60 cells was evaluated across the concentration range
of 0.0625, 0.125, 0.250, 0.500, 1.000, 2.000, and 4.000 mg mLꢁ1 in
triplicate for each group. The cytotoxicity of HepG2 cells was
evaluated across the concentration range of 0.094, 0.188, 0.375,
0.750, 1.500, 3.000, and 6.000 mg mLꢁ1 in triplicate for each
group. Absorbance was measured at 490 nm using a microplate
reader (US Bio-Tek, model ELX800MV).
2.7 Characterization of the Fe3O4@NH2-b-CD MNPs
FTIR (Thermo Nicolet 6700 ThermoFisher Scientic, USA)
spectral analysis of b-CD, 6-OTos-b-CD, 6-N3-b-CD, 6-NH2-b-CD,
Fe3O4@NH2-b-CD MNPs, and Fe3O4@NH2-b-CD@GA MNPs was
performed. X-ray diffraction analysis (XRD, MAC Science
MXP18AHF) of the samples (Fe3O4 MNPs, Fe3O4@NH2-b-CD
MNPs and Fe3O4@NH2-b-CD@GA MNPs) was performed using
an X-ray powder diffractometer (Bruker, Germany) equipped
with Cu Ka radiation. The physical appearance and nano-
particle size distribution were evaluated using a high-resolution
transmission electron microscopy (HRTEM) system (SU8200,
Hitachi TEM system; Hitachi High-Technologies Pte Ltd).
Solutions of Fe3O4@NH2-b-CD MNPs and Fe3O4@NH2-b-
CD@GA MNPs were diluted to optimal concentrations with
absolute alcohol and placed on a copper grid prior to the
analysis. Hysteresis curves of Fe3O4, Fe3O4@NH2-b-CD MNPs
and Fe3O4@NH2-b-CD@GA MNPs were determined between
ꢁ20–20 KOe at 300 K. Determination of the average particle
size, polydispersity index (PDI), and zeta potential of Fe3O4
MNPs, Fe3O4@NH2-b-CD MNPs, and Fe3O4@NH2-b-CD@GA
MNPs were performed using a Malvern particle size analyzer
(Zetasizer 3000HS; Malvern Instruments, ꢂUK). Each experiment
was performed in triplicate (n ¼ 3) at 25 C.
2.11 Vascular irritation
The inuence of Fe3O4@NH2-b-CD@GA MNP concentration
and ear-vein injection times on vascular irritation in rabbit were
tested.26 Here, 18 white New Zealand rabbits (weighing 2–2.5 kg)
were divided randomly into two groups. GA solution (dissolved
in normal saline) and Fe3O4@NH2-b-CD@GA MNPs were
administered to the right ear vein of the two groups of rabbits at
a dose of 4, 8, or 16 mg kgꢁ1, respectively. An equivalent volume
of normal saline was administrated as a reference via the le ear
vein. All the rabbits were treated once daily for 3 days and
examined for signs of irritation aer administration. The
excised tissue samples were xed in 4% paraformaldehyde,
then embedded in paraffin, and subsequently, cut into 5 mm
thick paraffin sections and placed on a glass slide. The tissues
were stained with hematoxylin and eosin (H&E). The stimu-
lating effect of GA on venous blood vessels aer being wrapped
with Fe3O4@NH2-b-CD was judged by observing the histopath-
ological changes in the ear veins under a microscope.
2.8 Drug entrapment efficiency and drug loading
Fe3O4@NH2-b-CD@GA MNPs (10.00 mg) was dissolved in 1 mL
methanol, and the unencapsulated GA was separated by ultra-
sonication for 20 min and supercentrifuged at 12 000 rpm. The
concentration of GA in the supernatant was determined by
HPLC.23 The encapsulation efficiency (EE%) and drug loading
(DL%) of the Fe3O4@NH2-b-CD@GA MNPs were determined by
the following equations:
2.12 Pharmacokinetic study
Ten SD rats (220 ꢀ 20 g, male) were divided randomly into two
groups (n ¼ 5) for GA and Fe3O4@NH2-b-CD@GA MNP intra-
EE% ¼ WE/WA ꢃ 100%
DL% ¼ WE/WL ꢃ 100%
venous administration at a dose of 20 mg kgꢁ1 27 Blood samples
.
(0.2 mL) were collected from the orbital plexus at specic time
intervals (0, 2, 5, 10, 20, 30, 40, 60, 80, 100, 120 min) and put into
where WE is the amount of encapsulated GA in the Fe3O4@NH2- heparin-containing tubes. Subsequently, the sample was
b-CD@GA MNPs, WA is the amount of GA added in the system, centrifuged at 3000 rpm for 10 min to separate plasma and then
ꢂ
and WL is the weight of Fe3O4@NH2-b-CD@GA MNPs added in stored at ꢁ80 C for further analysis. 10 mL of the internal
system.
standard (2 mg mLꢁ1 of gambogic acid) was spiked in 100 mL rat
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plasma, and subsequently, vortex-mixed with 1 mL ethyl acetate macromolecule to form the nano complex Fe3O4@NH2-b-
for 10 min. The supernatant was gathered aer being centri- CD@GA MNPs (Scheme 1).
fuged at 3500 rpm for 10 min and evaporated under nitrogen.
Next, 100 mL of methanol was added to the eppendorf tubes to
redissolve, and then the solution was centrifuged at 12 000 rpm;
20 mL of the supernatant was analyzed by HPLC.
4.1 Characterization of the Fe3O4@NH2-b-CD MNPs
The FTIR spectral analysis results of b-CD, 6-NH2-b-CD,
Fe3O4@NH2-b-CD MNPs, and Fe3O4@NH2-b-CD@GA MNPs are
shown in Fig. 1. The spectra of the composites are similar to
each other because they all contain infrared active bands orig-
inating from b-CD. Nonetheless, in the spectral range from 1300
to 1800 cmꢁ1 and below 800 cmꢁ1, certain differences can be
observed due to the presence of Fe3O4.28 b-CD contains multiple
hydroxyl groups, and their reactivity is different. The absorption
bands at 3387 cmꢁ1 correspond to OH or COOH stretching
vibration, and at 1015 and 1135 cmꢁ1 are related to the anti-
symmetric glycosidic na(C–O–C) vibration and the coupled n(C–
C/C–O) stretching vibration (Fig. 1a-a), respectively, which were,
according to the R-1, 4-bond skeleton vibration, and the anti-
symmetric vibrations of glycosidic bond (C–O–C).29 The peaks of
3. Statistical analysis
All the results are expressed as mean ꢀ standard deviation (SD)
and were calculated using GraphPad Prism 7.0. Data were
evaluated using the Student's t-test. P values less than 0.05 were
considered to show statistical difference and less than 0.01 was
considered to show signicant difference.
4. Results and discussion
The synthesis of 6-NH2-b-CD was achieved by substituting the 6- 6-NH2-b-CD at 1635 cmꢁ1 are related to the N–H stretching
position hydroxyl group of b-CD with p-TosCl, NaN3, and P(Ph)3 vibration (Fig. 1a-b). The results indicate that –NH2 was
via a reduction reaction. Subsequently, the carboxyl group (on successfully graed onto the b-CD.30 The signicant peak of
the citric acid) on the surface of Fe3O4 was activated by EDCI Fe3O4@NH2-b-CD was observed at 580 cmꢁ1, which was
and NHS to react with ammonia on 6-NH2-b-CD to form an ascribed to the Fe–O stretching vibration of Fe3O4 (Fig. 1a-c).
amide, and produced Fe3O4@NH2-b-CD MNPs as the host Signicant peaks of Fe3O4@NH2-b-CD@GA MNPs at 2950 cmꢁ1
macromolecule. The GA, which is a highly hydrophobic anti- observed were due to the C–H bond stretching vibration of citric
tumor drug, was the guest component included in the host acid.31 The absorption peaks at 1637 and 1015 cmꢁ1 (Fig. 1a-d)
Scheme 1 Schematic diagram of the synthesis process of Fe3O4@NH2-b-CD@GA MNPs.
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Fig. 1 Characterization of Fe3O4@NH2-b-CD@GA MNPs: (a). FTIR spectra of b-CD (a), 6-NH2-b-CD (b), Fe3O4@NH2-b-CD MNPs (c) and
Fe3O4@NH2-b-CD@GA MNPs (d) and (b). The XRD of Fe3O4 MNPs (a), Fe3O4@NH2-b-CD MNPs (b) and Fe3O4@NH2-b-CD@GA MNPs (c).
conformed to the glucoside bond (C–O–C) anti-symmetric immediately aer the production by using a Zetasizer instru-
vibrations and coupled vibrations (C–C/C–O) of GA, which ment (Fig. 3). The average particle sizes of Fe3O4@NH2-b-CD
suggested that GA was loaded onto the Fe3O4@NH2-b-CD MNPs. MNPs and Fe3O4@NH2-b-CD@GA MNPs were 119.4 ꢀ 0.37 nm
All the changes indicated that the preparation of Fe3O4@NH2-b- and 147.4 ꢀ 0.28 nm and their polydispersity indexes (PDIs)
CD@GA was successful.
were 0.092 ꢀ 0.025 and 0.072 ꢀ 0.013, respectively. The zeta
Fig. 1b shows the XRD spectral analysis of the products potentials of Fe3O4@NH2-b-CD MNPs and Fe3O4@NH2-b-
Fe3O4 MNPs, Fe3O4@NH2-b-CD MNPs, and Fe3O4@NH2-b- CD@GA MNPs were ꢁ30.8 ꢀ 0.16 mV and ꢁ29.3 ꢀ 0.42 mV,
CD@GA MNPs. Fig. 1b-a show the XRD pattern of the Fe3O4 respectively. These results show that the particle size of
MNPs. Compared with the standard XRD pattern of Fe3O4 Fe3O4@NH2-b-CD MNPs increased aer loading GA. According
(JCPDS le 79-0417, magnetite), the observed reections at to the report, nanoparticles with a positive surface charge are
30.1ꢂ, 35.4ꢂ, 43.1ꢂ, 56.9ꢂ, 62.8ꢂ were identied.32 For prone to interact non-specically with serum proteins in blood
Fe3O4@NH2-b-CD@GA MNPs, the observed reections at (30.2ꢂ, and produce precipitation. The negative zeta potential of the
220), (35.5ꢂ, 311), (43.2ꢂ, 400), (53.7ꢂ, 422), (57.2ꢂ, 511), and nanoparticles suggested that they could have a prolonged
(62.7ꢂ, 440) were identied. For Fe3O4 MNPs (Fig. 1b-b) and circulation time in blood. The zeta potential of Fe3O4@NH2-b-
Fe3O4@NH2-b-CD@GA MNPs (Fig. 1b-c), the characteristic CD@GA MNPs was about ꢁ29.3 mV, which meant they were not
peaks of Fe3O4 MNPs were always present. The results indicated dispersed stably as the electrostatic repulsion could not prevent
that the modication did not change the crystal structure of the the colloids from occulation.
Fe3O4 MNPs and the Fe3O4@NH2-b-CD@GA MNPs were
successfully prepared.
The magnetic properties of Fe3O4 MNPs, Fe3O4@NH2-b-CD
MNPs, and Fe3O4@NH2-b-CD@GA MNPs were investigated by
The crystallographic structures of the Fe3O4@NH2-b-CD@GA VSM (Vibrating Sample Magnetometer, NanoMagnetics Instru-
MNPs and Fe3O4@NH2-b-CD@GA MNPs were analyzed using ments) at 300 K. Their hysteresis loops could be clearly observed
HRTEM (Fig. 2). The images in Fig. 2a1 and b1 show that the in Fig. 4. In the S-shape magnetic curve, no coercivity and
Fe3O4@NH2-b-CD MNPs and Fe3O4@NH2-b-CD@GA MNPs remnant magnetization were observed, suggesting that samples
have a substantially spherical-like core–shell structure, where were superparamagnetic.35 Superparamagnetic materials do not
the core is Fe3O4 (black region) and the shell is 6-NH2-b-CD retain magnetization before and aer exposure to an external
(gray region), conrming the direct deposition of a 6-NH2-b-CD magnetic eld, which is very useful in in vivo applications. The
particle layer on the surface of Fe3O4 MNPs, and that no saturation magnetization values for Fe3O4, Fe3O4@NH2-b-CD
amorphous 6-NH2-b-CD was observed. The circular streak MNPs, and Fe3O4@NH2-b-CD@GA MNPs were 56.86, 53.64, and
image in Fig. 2a2 reveals the orientation of the crystalline Fe3O4 41.75 emu gꢁ1, respectively. The saturation magnetization value
core, and the diameter spacing of 0.27 nm, which agrees with of Fe3O4@NH2-b-CD@GA MNPs was considered to be sufficient
that of the standard magnetite of Fe3O4.33 Another diameter for drug delivery. The decrease in the saturation magnetization
spacing of 0.47 nm was measured in the shell layer, which could value was attributed to the non-magnetic b-CD on the particles,
be ascribed to the amorphous Fe3O4 phase,34 as shown in quenching the magnetic moment.
Fig. 2a2. The electron diffraction pattern exhibits spotty
diffraction rings and well-resolved spots in Fig. 2a3, which have the size effect and strong adsorption capacity of magnetic
conrm the crystalline structure of the nanoparticles. nanoparticles, but also have a hydrophobic and hydrophilic
As a drug-loading material, Fe3O4@NH2-b-CD MNPs not only
The size distribution and zeta potential of Fe3O4@NH2-b-CD cavity inside the cyclodextrin. The GA content in the
MNPs and Fe3O4@NH2-b-CD@GA MNPs were determined Fe3O4@NH2-b-CD@GA MNPs was measured using HPLC. The
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Fig. 2 The HRTEM image of Fe3O4@NH2-b-CD MNPs (a1, a2, a3) and Fe3O4@NH2-b-CD@GA MNPs (b1, b2, b3).
EE% and DL% of the GA in the Fe3O4@NH2-b-CD@GA MNPs b-CD. When the reaction temperature exceeds 10 ꢂC and the
NaOH concentration is too high, the resulting 6-OTs-b-CD can
were 85.71 ꢀ 3.47% and 4.63 ꢀ 0.04%, respectively.
During the preparation of 6-NH2-b-CD, b-CD was prone to be easily hydrolyzed. There are two main reactions for the
clathration, whereas it was difficult to dissolve TosCl in water, formation of an azide group: one is Pd/C reduction, and the
and the whole reaction system was heterogeneous. Therefore, other is triphenylphosphine reduction. The Pd/C reduction was
using water as a solvent can effectively inhibit the clathration of more intense and produces more by-products. The reduction
Fig. 3 The size distribution and zeta potential distribution of Fe3O4@NH2-b-CD MNPs and Fe3O4@NH2-b-CD@GA MNPs (n ¼ 3).
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Fig. 4 The saturation magnetization curve of Fe3O4 MNPs, Fe3O4@NH2-b-CD MNPs, and Fe3O4@NH2-b-CD@GA MNPs.
reaction of triphenylphosphine was milder, and the structure of CD, the magnetic strength of the magnetic center was
the phosphorimide could be obtained, while the use of acetone weakened.
was effective for preventing the re-dissolution of triphenyl-
phosphine in aqueous ammonia.
4.2 In vitro drug release
In the synthesis of Fe3O4 MNPs, stirring was carried out
The suitability of Fe3O4@NH2-b-CD MNPs as drug carriers was
using polytetrauoroethylene. Citric acid is a polycarboxyl
evaluated using cumulative drug release experiments. Fig. 5
molecule that may be adsorbed by Fe3O4 during the synthesis
shows the release percent of GA released from Fe3O4@NH2-b-
CD@GA MNPs under different pH conditions. The results
revealed that Fe3O4@NH2-b-CD@GA MNPs showed the highest
process, which can reduce the surface energy of the molecule,
can effectively prevent the occurrence of agglomeration, and
also act as a dispersant or electrostatic stabilizer in the
release efficiency, where the cumulative release percent was
synthesis process.36 The b-CD was modied into 6-NH2-b-CD,
nearly 30% within 100 min under pH 7.4. The cumulative
release of GA was lower at pH 4.0. However, in subsequent drug
and its action was more inclined to provide –NH2, which formed
a chemical bond with –COOH and was graed onto Fe3O4.37 In
releases, the difference was not obvious under different pH
addition, 6-NH2-b-CD can also be used as an inclusion material,
conditions. As Fig. 5 indicates, the GA release from Fe3O4@NH2-
b-CD@GA MNPs constituted three different phases: an initial
whereby the hydrophobic cavity can load a poorly soluble drug
and act as a drug carrier. Fe3O4 has one molecule of FeO with
sp2 hybridization and one molecule of Fe2O3 with sp3 hybrid-
relatively fast phase, an acceleration phase, and a smooth
release phase, which would help in sustaining the drug to be
ization, according to the principle of Pauli [exclusion] prin-
available for a longer period of time and helping reduce the
ciple.38 Fe3O4 can provide an empty orbital to chelate with acid
toxic side effects of the drugs.
ions. Furthermore, as the Fe3O4 forms nanoparticles, the
surface energy and adsorption capacity were greatly increased,
and the free acid ions were adsorbed on the surface of the Fe3O4
in the solution. Finally, Fe3O4–COOH was formed.
4.3 Cytotoxicity analysis
In an attempt to understand the anticancer efficacy of
Fe3O4@NH2-b-CD@GA MNPs in vitro, APL HL-60 and HepG2
cells, being two different cancer cell lines, were treated with
different concentrations of GA, Fe3O4@NH2-b-CD, and
Fe3O4@NH2-b-CD@GA MNPs for 24 h. The MTT assay showed
that GA was able to inhibit the growth of HL-60 cells and HepG2
There are three main reasons for the decrease in saturation
magnetization. First, the original spin state of the ferroferric
oxide has changed. Second, with the nanocrystallization of
Fe3O4, non-collinear rotation occurs, resulting in a decrease in
the magnetic moment and magnetic properties, or rather it can
be said that the surface curvature of the particle is affected by
the size, and this changes the disordered crystal orientation of
the particle surface, resulting in a change in saturation
magnetization. Third, aer Fe3O4 was encapsulated by NH2-b-
cells, with IC50 values of 0.818 mg mLꢁ1 and 1.525 mg mLꢁ1
,
respectively. Additionally, as observed from the decrease in
corresponding IC50 values (0.348 and 0.964 mg mLꢁ1 in HL-60
and HepG2 cells, respectively) in the two cancer cell lines,
aer GA was encapsulated into Fe3O4@NH2-b-CD, the
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Fig. 5 Accumulative release of GA and Fe3O4@NH2-b-CD@GA MNPs over time under different pH conditions (n ¼ 3).
antiproliferative efficacy was greatly augmented. Furthermore, necrosis, and formation of thrombus when the dose of injection
in HL-60 and HepG2 cells, Fe3O4@NH2-b-CD@GA MNPs was increased. No pathological change was found in the control
compared to GA at an equivalent drug concentration showed group (Fig. 7a–c). There was no degeneration, necrosis, and
higher cytotoxicity, while Fe3O4@NH2-b-CD demonstrated no endothelial cell injury with the inner wall of veins, and a slight
obvious cytotoxicity, indicating its good safety with the cells, as swelling in Fe3O4@NH2-b-CD@GA MNP groups was observed,
shown in Fig. 6.
as shown in Fig. 7g–i. This suggested that the vascular irritation
of GA could be reduced with Fe3O4@NH2-b-CD MNP encapsu-
lation due to the reduction of the direct contact of the drug with
the vascular endothelium. Therefore, it could be acceptable for
intravenous administration.
4.4 Vascular irritability
Macroscopic observations were performed for a single ear
injection of GA and Fe3O4@NH2-b-CD@GA MNPs to rabbits. In
the GA group, signicant redness, congestion, and agglomera-
tion occurred in the injection area of the rabbit ear,39 but no
4.5 Pharmacokinetic characteristics
such phenomena occurred in the Fe3O4@NH2-b-CD@GA MNPs The pharmacokinetic characteristics were evaluated using i.v.
group. As presented in Fig. 7d–f, there was signicant swelling, administration at a single dose of 20 mg kgꢁ1 of GA and
degeneration, vascular edema, inammatory cell inltration, Fe3O4@NH2-b-CD@GA MNPs in rats, respectively. The
Fig. 6 Effects of GA, Fe3O4@NH2-b-CD MNPs, and Fe3O4@NH2-b-CD@GA MNPs on the cell viability of (a) HL-60; (b) HepG2. Cells in 96-well
plates were treated with various concentrations of GA, Fe3O4@NH2-b-CD MNPs, and Fe3O4@NH2-b-CD@GA MNPs for 24 h (mean ꢀ SD, n ¼
3).*p < 0.05, **p < 0.01 compared with the Fe3O4@NH2-b-CD MNPs, #p < 0.05, ##p < 0.01 compared with the GA.
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Fig. 7 Pathological paraffin sections (hematoxylin-eosin stain) from the ears of rabbits (ꢃ200 magnification). Control group (a–c), GA group (d–
f), Fe3O4@NH2-b-CD@GA MNPs (g–i).
pharmacokinetic parameters were calculated using DAS 2.1 group (p < 0.01). In addition, the t1/2 value of Fe3O4@NH2-b-
soware and the noncompartment model. The pharmacoki- CD@GA MNPs was signicantly improved to 18.823
ꢀ
netic parameters are listed in Table 1, respectively, and the 0.409 min compared with that of the GA group. These results
concentration-time proles of GA and Fe3O4@NH2-b-CD@GA suggested that Fe3O4@NH2-b-CD@GA MNPs could overcome
MNPs are presented in Fig. 8. Encapsulating GA in Fe3O4@NH2- the low bioavailability and poor pharmacokinetics of GA. The in
b-CD greatly increased the systemic drug exposure; the AUC
increased from (149.2 ꢀ 11.91 mg (L min)ꢁ1) to (347.8 ꢀ
13.71 mg (L min)ꢁ1), and the Cmax value in the Fe3O4@NH2-b-
CD@GA MNPs group was signicantly higher than in the GA
Table
1 Pharmacokinetic parameters of GA in plasma following
administration of GA and Fe3O4@NH2-b-CD@GA MNPs to rats (mean
ꢀ SD, n ¼ 6)
Parameters
GA
Fe3O4@NH2-b-CD@GA
t1/2z (min)
6.932 ꢀ 0.392
18.823 ꢀ 0.409a
11.82 ꢀ 0.27b
Cmax (mg Lꢁ1
)
7.54 ꢀ 0.56
0.150 ꢀ 0.004
149.2 ꢀ 11.76
199.968 ꢀ 15.36
CLz (L (min kg)ꢁ1
)
0.062 ꢀ 0.024b
347.801 ꢀ 13.72b
414.707 ꢀ 14.62a
AUC(0–t) (mg (L min)ꢁ1
)
AUC(0–N) (mg (L min)ꢁ1
)
Fig. 8 Mean concentration-time profile of GA and Fe3O4@NH2-b-
CD@GA MNPs in plasma following i.v. administration of a single dose
of 20 mg kgꢁ1 to rats (mean ꢀ SD, n ¼ 6).
a
p < 0.05. b p < 0.01 compared with the GA group.
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vivo pharmacokinetic study proved that the Fe3O4@NH2-b-
CD@GA MNPs exhibited slower clearance and higher drug
exposure than GA did. The reason for this is that b-CD them-
selves have a sustained release feature, and the other reason is
that the aminated modication increases this characteristic.40
Besides, the concentration–time characteristic curve in vivo did
not exactly match the in vitro release prole. The amount of
released GA over 12 h was approximately 67% in the GA group,
while the half-life was approximately 6.9 min aer i.v. admin-
istration to the rats, which corresponded to a release amount of
about 4.8% in vitro. The amount of released GA over 12 h was
75% in the Fe3O4@NH2-b-CD@GA MNPs group in vitro, while
the half-life was approximately 18.8 min aer i.v. administra-
tion to rats in vivo. Some studies suggest that changes in the
morphology and surface electronic structure of nanoparticles
may have an effect on the pharmacokinetics in vivo.41 These
results provided us some information that Fe3O4@NH2-b-CD
themselves may have a sustained release feature to understand
the properties of Fe3O4@NH2-b-CD@GA MNPs with respect to
pharmacokinetics and pharmacodynamics.
Acknowledgements
The project was supported by a grant from National Natural
Science Foundation of China (No. 81403318), Nature and
Science Foundation of Department of Education, Anhui prov-
ince in China (No. KJ2014A134), Undergraduate Training
Projects for Innovation and Entrepreneurship, Anhui province
in China (No. 2016150) and the Key project of Department of
Scientic Technology, Anhui province in China (1604f0804030)
Author. All animal procedures were performed in accordance
with the Guidelines for Care and Use of Laboratory Animals of
Anhui University of Chinese Medicine and experiments were
approved by the Animal Ethics Committee of Anhui University
of Chinese Medicine.
References
1 S. Rajput and M. Mandal, Eur. J. Cancer Prev., 2012, 21, 205–
215.
Fe3O4@NH2-b-CD@GA MNPs possessed better magnetic
targeting, controlled release, low stimulation, and good phar-
macokinetic behavior in vivo, including a lower CLz, longer half-
life, and increased AUC, which indicate that they have a good
promoting effect on enhancing drug accumulation and
adsorption at the tumor site when compared with gambogic
acid nanomagnetic systems, such as gambogic acid-loaded
micelles based on chitosan derivatives,42 magnetic nano-
particles of Fe3O4 with gambogic acid,43 GO-modied Fe3O4/
SiO2 nanoparticles with combined rhenium-188 and gambogic
acid,44 and gambogic acid-loaded magnetic Fe3O4 nano-
particles.45 Moreover, other nanomagnetic systems, such as
pegylated liposomes loaded with cisplatin magnetic nano-
particles (Cis-MLs),46 and folic acid-functionalized magnetic
nanoparticles47 also have great signicance.
2 G. M. Huang, Y. Sun, X. Ge, et al., World J. Gastroenterol.,
2015, 21, 6194–6205.
3 Q. Guo, Q. You, Z. Wu, et al., Acta Pharmacol. Sin., 2004, 25,
769–774.
4 F. Liu, X. Huang, L. Han, M. Sang, L. Hu, B. Liu, B. Duan,
P. Jiang, X. Wang, Z. Qiao, C. Ma, W. Liu, J. Liu, F. Feng
and W. Qu, Biomater. Sci., 2019, 7, 1028–1042.
5 X. Tang, J. Sun, T. Ge, K. Zhang, Q. Gui, S. Zhang and
W. Chen, Colloids Surf., B, 2018, 172, 26–36.
6 S. Zhang, Q. Li, L. Zhang, H. Sun and Q. You, Chin. J. Org.
Chem., 2012, 32, 1450–1458.
7 B. Andrzejewski, W. Bednarski, M. Ka´zmierczak, et al.,
Composites, Part B, 2014, 64, 147–154.
8 Q. A. Pankhurst, J. Connolly, S. K. Jones, et al., J. Phys. D:
Appl. Phys., 2003, 36, R167–R181.
9 O. Veiseh, J. W. Gunn and M. Zhang, Adv. Drug Delivery Rev.,
2010, 62, 284–304.
10 C. Sun, J. Lee and M. Zhang, Adv. Drug Delivery Rev., 2008, 60,
1252–1265.
5. Conclusions
Fe3O4@NH2-b-CD@GA MNPs based on the host macromolecule 11 H. Mittal, V. Parashar, S. B. Mishra and A. K. Mishra, Chem.
Fe3O4@NH2-b-CD and the guest component GA showed Eng. J., 2014, 255, 471–482.
a substantially spherical-like core–shell structure with an 12 J. L. Gong, B. Wang, G. M. Zeng, C. P. Yang, C. G. Niu,
average size of 147.4 nm and zeta potential of ꢁ29.3 mV.
Q. Y. Niu, W. J. Zhou and Y. Liang, J. Hazard. Mater., 2009,
164, 1517–1522.
Besides, the Fe3O4@NH2-b-CD@GA MNPs showed enhanced
stability, sustained drug release proles, and essentially 13 R. A. Frimpong and J. Z. Hilt, Nanomedicine, 2010, 5, 1401–
increased cytotoxicity compared with free GA. Moreover, 1414.
Fe3O4@NH2-b-CD@GA MNPs could extend the biological half- 14 V. D. M. Frank, T. Vermonden, C. F. Van Nostrum, et al.,
life and improve the vascular irritability and bioavailability Biomacromolecules, 2009, 10, 3157–3175.
compared with GA. Collectively, the present results suggested 15 T. Losson and D. Duchene, Int. J. Pharm., 2007, 329, 1–11.
¨
that the encapsulation of GA could greatly enhance the bio- 16 R. Holm, C. Schonbeck, P. Somprasirt, P. Westh and H. Mu,
logical activity, which also would provide more choices for
Fe3O4@NH2-b-CD as carriers for other hydrophobic drugs.
J. Inclusion Phenom. Macrocyclic Chem., 2014, 80, 243–251.
17 C. E. Jensen, R. A. dos Santos, A. M. Denadai, C. F. Santos,
A. N. Braga and R. D. Sinisterra, Molecules, 2010, 15, 4067–
4084.
18 L. Huang, H. Wang, B. Li, E. Li, Y. Zhou, Y. Yang, C. Dong
and S. Shuang, J. Inclusion Phenom. Macrocyclic Chem.,
2014, 80, 209–215.
Conflicts of interest
There are no conicts to declare.
This journal is © The Royal Society of Chemistry 2019
RSC Adv., 2019, 9, 27136–27146 | 27145

View Article Online
RSC Advances
Paper
19 P. Huang, L. L. Yang, C. Y. Wang, J. P. Chen, S. S. Wang, 33 E. Liu, H. Yuan, Z. Kou, X. Wu, Q. Xu, Y. Zhai, Y. Sui, B. You,
S. J. Wang, Y. J. Chen, D. L. Wang and H. P. Huang, J.
Nanosci. Nanotechnol., 2015, 15, 4774–4783.
20 H. Li, M. H. El-Dakdouki, D. C. Zhu, G. S. Abela and
X. Huang, Chem. Commun., 2012, 48, 3385–3387.
21 I. A. Anan'Eva, E. N. Myshak, E. N. Shapovalova, et al., J. Anal.
Chem., 2003, 58, 461–466.
J. Du and H. Zhai, Sci. Rep., 2015, 5, 11164–11173.
34 Z. C. Xu, Y. L. Hou and S. H. Sun, J. Am. Chem. Soc., 2007, 129,
8698–8699.
35 H. Ebrahimzadeh, E. Moazzen, M. M. Amini and O. Sadeghi,
Anal. Methods, 2012, 4, 3232–3237.
36 M. Namvari and H. Namazi, Polym. Int., 2014, 63, 1881–1888.
22 S. A. Kulkarni, P. S. Sawadh, K. K. Kokate, et al., Foundation of 37 L. Fan, C. Luo, M. Sun, H. Qiu and X. Li, Colloids Surf., B,
Computer Science (FCS), 2012. 2013, 103, 601–607.
23 M. Filippa, M. I. Sancho and E. Gasull, J. Pharm. Biomed. 38 F. Tennie, V. Vedral and C. Schilling, Phys. Rev. A, 2017, 95,
Anal., 2008, 48, 969–973. 022336–022345.
24 Z. Tao, Y. Zhou, J. Lu, W. Duan, X. He, L. Lin, J. Ding and 39 R. Li, Y. Chen, F. Zhao, Y. Liu, L. Wen and L.-l. Zeng, Chin. J.
Y. Qin, Cancer Biol. Ther., 2014, 6, 691–696. Oncol., 2009, 31, 810–814.
25 R. Mu, N. Lu, J. Wang, Y. Yin, Y. Ding, X. Zhang, H. Gui, 40 S. S. Banerjee and D.-H. Chen, J. Nanopart. Res., 2008, 11,
Q. Sun, H. Duan, L. Zhang, Y. Zhang, X. Ke and Q. Guo,
Eur. J. Cancer Prev., 2010, 19, 61–67.
26 N. Lu, Y. Yang, Q.-D. You, Y. Ling, Y. Gao, H.-Y. Gu, L. Zhao,
X.-T. Wang and Q.-L. Guo, Cancer Lett., 2007, 258, 80–89.
27 K. Hao, X.-P. Zhao, X.-Q. Liu and G.-J. Wang, Biomed.
Chromatogr., 2007, 21, 279–283.
28 S. Kaamyabi, D. Habibi and M. M. Amini, Bioorg. Med. Chem.
Lett., 2016, 26, 2349–2354.
29 T. Losson and M. Masson, Int. J. Pharm., 2001, 225, 15–30.
2071–2078.
41 M. Hadjidemetriou, Z. Al-Ahmadi, M. Mazza, et al., ACS
Nano, 2015, 9, 8142–8156.
42 X. Zhu, C. Zhang, X. Wu, et al., Drug Dev. Ind. Pharm., 2008,
34, 2–9.
43 B. Chen, Y. Liang, W. Wu, J. Cheng, et al., Int. J. Nanomed.,
2009, 4, 251–259.
44 Y. Yang, Y. Liu, C. Cheng, et al., ACS Appl. Mater. Interfaces,
2017, 9, 28195–28208.
30 E. Sikorska, M. Dawgul, K. Greber, E. Ilowska, A. Pogorzelska 45 C. Wang, H. Zhang, Y. Chen and B. Chen, Int. J. Nanomed.,
and W. Kamysz, Biochim. Biophys. Acta, 2014, 1838, 2625–
2012, 7, 781–787.
2634.
46 A. Toro-Cordova, M. Flores-Cruz, J. Santoyo-Salazar, et al.,
Molecules, 2018, 23, 2272–2288.
¨
31 G. Trettenhahn and A. Koberl, Electrochim. Acta, 2007, 52,
2716–2722.
47 K. Niemirowicz, H. Car, A. Sadowska, et al., J. Biomed.
Nanotechnol., 2017, 13, 665–677.
32 A. Tauq, Sunaryono, E. G. Rachman Putra, et al., Mater. Sci.
Forum, 2015, 827, 213–218.
27146 | RSC Adv., 2019, 9, 27136–27146
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