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ture vigorously stirred for 4 h. The reaction mixture was allowed to
stand until completely stratified, whereby the brown amino-func-
tionalized MNPs were transferred into the upper aqueous layer.
The hydrophilic MNPs were separated and then isolated by centri-
fugation and washing with water and ethanol (yield: 74–77%). The
obtained products were dispersed in H2O. The amount of amino
groups on the surface of MNPs was determined by using the stan-
dard Fmoc quantification protocol.[17]
Experimental Section
Materials
Iron(III) trichloride, manganese(II) chloride tetrahydrate, ethanol,
hexane, diphenyl ether, and folic acid (FA) were purchased from
Sinopharm Chemical Reagent (Shanghai, China). 6-Aminohexanoic
acid (AHA), oleylamine, and 9-fluorenylmethyl chloroformate
(Fmoc-Cl) were purchased from J&K Chemical (Shanghai, China).
Rhodamine B isothiocyanate (RBITC), N-hydroxysulfosuccinimide
(Sulfo-NHS), 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydro-
chloride (EDC), and hydrazine hydrate solution (78–82%) were pur-
chased from Sigma-Aldrich (Shanghai, China). All reagents were
used without further purification. Water used in all experiments
was purified by using a Milli-Q Plus 185 water purification system
(Millipore, Bedford, MA) to give a resistivity higher than 18 MWcm.
Synthesis of rhodamine B and FA conjugates of Fe3O4 and
Mn3O4
To modify the MNPs-NH2 with a fluorescent dye, MNPs-NH2 (3.6ꢂ
10À3 mmol NH2, ca. 30 mg Fe3O4 or 32 mg Mn3O4) was dissolved in
100 mL of dried THF, and RBITC (1.0 mg, molar ratio of RBITC/
NH2 =1/2) was added to the solution. The mixture were stirred
under nitrogen atmosphere in the dark at room temperature for
5 h. The resulting MNP(RB)-NH2 was precipitated by centrifugation
at 12000 rpm for 10 min and purified by several cycles of redisper-
sion in ethanol and centrifugation. Then, FA was further conjugat-
ed with the remaining amino-groups of MNP(RB)-NH2. Briefly, FA
(8.0 mg, 1.8ꢂ10À2 mmol, molar ratio of FA/NH2 =10/1) was dis-
solved in 20 mL of anhydrous DMSO, EDC and Sulfo-NHS (molar
ratio FA/EDC/Sulfo-NHS=1/2/2.5) were added, and the mixture
was stirred gently at room temperature for 3 h. 30 mg of MNP(RB)-
NH2 dissolved in 10 mL of DMSO was added to the activated FA so-
lution, and the mixture stirred gently for another 3 h at room tem-
perature. The final MNP(RB)-FA was separated by centrifugation at
12000 rpm for 10 min and washed three times with phosphate-
buffered saline (PBS, pH 7.4). The product was dispersed in PBS
and stored at 48C.
Synthesis of the Phth-protected metal–organic precursor
Firstly, the Phth-protected ligand was synthesized by dehydrative
condensation of 6-aminohexanoic acid with phthalic anhydride
(92.4% yield). A mixture of 6-aminohexanoic acid and phthalic an-
hydride in 1:1 molar ratio was heated at 1708C for 4 h. After the
mixture was cooled to room temperature, a white powder was fil-
tered from the reaction medium, washed with deionized water,
and dried in vacuum. Then, the Phth-protected metal–organic pre-
cursor was prepared by the reaction of metal ions with the Phth-
protected ligand. Briefly, 3 mmol of MnCl2·4H2O with 6 mmol of
Phth-protected ligand or 3 mmol of FeCl3·6H2O with 9 mmol of
Phth-protected ligand were dissolved in 25 mL of CH2Cl2, 2.5 mL of
triethylamine was added dropwise, and the reaction mixture was
stirred for 5 h. The final product was washed three times with de-
ionized water to remove residual metal ions, the solvent was re-
moved on a rotary evaporator, and the dark brown precursor were
obtained (ca. 96% yield).
Cell culture
A human cervical carcinoma cell line (HeLa) and a human breast
adenocarcinoma cell line (MCF-7) were provided by Shanghai Insti-
tutes for Biological Sciences (SIBS), Chinese Academy of Sciences
(CAS, China). Cells were cultured in Dulbecco’s modified Eagle’s
medium (DMEM) supplemented with 10% fetal bovine serum (FBS)
at 378C and 5% CO2. Cells were plated in tissue culture flasks
under 100% humidity.
Synthesis of amino-functionalized MNPs
The Phth-protected metal–organic precursor (12 mmol) was dis-
solved in in 20 mL of diphenyl ether/oleylamine in a 100 mL three-
neck flask. The Mn3O4 NPs were synthesized by two-stage heating:
first, the mixture was rapidly heated to 1208C and held at that
temperature for 1 h to remove the water from the solution;
second, the reaction temperature was increased to 1608C at a heat-
ing rate of 58CminÀ1 and maintained at that temperature for 4.5 h
under a flow of nitrogen. The MnFe2O4 and Fe3O4 NPs were synthe-
sized by three-stage heating: first, the reaction solution was rapidly
heated to 1208C and held at that temperature for 1 h to remove
the water; second, the mixture was heated to 2108C at a heating
rate of 58CminÀ1 and maintained at that temperature for 2 h;
third, the reaction temperature was further increased to 2608C and
kept at that temperature for 1.5 h. Diphenyl ether/oleylamine
volume ratios of 20/0, 18/2, and 15/ 5 were used for the synthesis
of Mn3O4, MnFe2O4, and Fe3O4 MNPs, respectively. The final reaction
solution was cooled to room temperature, and the black-brown
MNPs were isolated by adding an excess of ethanol followed by
centrifugation and washing with ethanol and hexane three times
(yield: 57–66% based on metal). The Phth-protected hydrophobic
MNPs (Phth-MNPs) can be easily dispersed in nonpolar organic sol-
vents such as hexane, toluene, and dichloromethane. The amino-
functionalized MNPs were obtained by deprotection of the Phth-
MNPs with hydrazine. Phth-MNPs were dispersed in 15 mL of
CH2Cl2, a mixture of 15 mL of H2O, 15 mL of EtOH, and 15 mL of
hydrazine hydrate solution (82%) was added, and the reaction mix-
In vitro cytotoxicity assay
In vitro cytotoxicity of Fe3O4(RB)-FA and Mn3O4(RB)-FA was evaluat-
ed by MTT assay of HeLa and MCF-7 cells. Cells were seeded into
a 96-well cell culture plates at 5ꢂ104 cells per well in DMEM with
10% FBS at 378C and 5% CO2 for 24 h; then the cells were incu-
bated with Fe3O4(RB)-FA (or Mn3O4(RB)-FA) with different Fe (or
Mn) concentrations (0, 10, 25, 50, 75, 100 mgmLÀ1 diluted in
DMEM) for 12 h or 24 h at 378C under 5% CO2, respectively. There-
after, MTT (10 mL, 5 mgmLÀ1) was added to each well, and the
plate was incubated for 4 h at 378C. After addition of 10% sodium
dodecyl sulfate (SDS, 100 mL/well), the OD570 value (Abs.) of each
well with background subtraction at 690 nm was measured on
Thermo Multiskan MK3-based microplate reader. The following for-
mula was used to calculate the inhibition of cell growth: cell viabil-
ity [%]=(mean of Abs. value of treatment group/mean Abs. value
of control)ꢂ100.
In vitro MRI
HeLa and MCF-7 cells (5ꢂ106) were separately incubated with
Fe3O4(RB)-FA (or Mn3O4(RB)-FA) with different Fe (or Mn) concentra-
Chem. Eur. J. 2014, 20, 7160 – 7167
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