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Guo et al.
The c(RGDyK)-Conjugated Fe3O4 Nanoparticles
improving cellular and nuclear drug uptake in tumors. Vari-
ous targeting ligands, such as peptides, antibodies, proteins
or small molecules have been coupled to the nanoparticle
carriers for increasing drug deliver efficient.12–14 By incor-
porating the designs of environment response mechanisms
in drug carrier, such as acidic pH or enzymatic cleavage in
the endosomes/lysosomes, anticancer drugs can be released
intracellularly and targeted to specific organelles.15ꢀ16
Integrin ꢁvꢂ3 plays an important role in tumor angio-
genesis and metastasis.17 It is upregulated in invasive
tumor cells, such as glioblastoma, melanoma, breast, ovar-
ian, and prostate cancers, but not in quiescent endothelium
and normal tissues.18–19 Its expression on carcinoma cells
potentiates metastasis by facilitating invasion and move-
ment across blood vessels. The cyclized RGD peptide
c(RGDyK) has been proven to target specifically to differ-
ent tumor cells expressing cell adhesion molecule integrin
ꢁvꢂ3.13ꢀ20 Suitably modified with cyclized RGD peptide
provides a special means for nanocarriers entering into the
cells.
spectrum was measured by an IFS-66V FT-IR spec-
trometer (Bruker Optics, Germany). Transmission electron
micrographs of the products were captured on a JEOL
Model JEM-200CX (Tokyo, Japan). Size analysis was per-
formed by a BI-SM200 dynamic light scattering instru-
ment (BrookHaven Instruments Corp., Long Island, USA).
Magnetic measurements were acquired using a vibrat-
ing sample magnetometer from Quautum Design MPMS-
XL7 SQUID Magnetometer (USA). The zeta potential
was obtained by a NANO-Z zeta potential instrument
(Malvern, USA.). The fluorescent images were measured
by a Zeiss LSM 710 fluorescent microscope (German).
Flow cytometry was carried out by a BD FACSCANT-
OTM Flow Cytometer (BD company, USA).
2.2. Synthesize of N-phthaloyl-L-glutamic
Acid Anhydride (GA)
0.025 mol phthalic anhydride and 0.025 mol L-glutamic
acid were mixed in a tube and heated up to liquate and
then for 15 min reaction. After cooling, the product was
recrystallized by ethanol-aqueous solutions and dried at
Here, we developed a drug delivery system with com-
bined three advantages: magnetic targeting, integrin ꢁvꢂ3
targeting and high drug loading. In this system, a carboxyl-
together with protected amino group-modified dual func-
tional Fe3O4 nano particle (DMP) has been synthesized
as carriers. c(RGDyK) is chemical coupled to the carrier
for special integrin ꢁvꢂ3-targeting. Doxorubicin (DOX),
ꢀ
40 C in vacuum for 24 h. Then 2.13 mL acetic anhydride
was added to the product obtained above and reflux for
15 min, the product GA was obtained after recrystallized
ꢀ
and dried at 40 C in vacuum for 24 h.
2.3. Generating of DMP
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which has a broad spectrum of antitumor properties in
IP: 79.110.18.19 On: Sun, 05 Jun 2016 01:53:53
Superparamagnetic nanoparticles (Fe3O4ꢃ was obtained by
therapy, was used as anticancer agents. By using a novel
Copyright: American Scientific Publishers
the coprecipitation of FeCl2 and FeCl3 under basic con-
multi-hand cross-linker poly-L-glutamic acid (PLGA),
DOX can be efficiently coupled to the carrier, and an effi-
cient approach for generating c(RGDyK)-modified Fe3O4
nanoparticles with high DOX load (R-DMP) for dual-
targeting integrin ꢁvꢂ3- expressing tumor cells has been
developed.
ditions as reported by Stroeve et al.21 The particle sur-
face was then transformed to SiO2 shell covered by a
sol–gel process22 using tetraethyl orthosilicate (TEOS).
100 mg obtained product was suspended in absolute
ethanol curtaining ammonia solution. Then 200 mg GA
and 3-aminopropyltrimethoxysilane (APS) were added
respectively. After the magnetite suspension was sonicated
5 min, the mixture was stirred for at 40 ꢀC for 12 h.
After ꢀwashed with absolute ethanol three times and dried
at 40 C in vacuum, the DMP product was obtained.
2. MATERIALS AND METHODS
2.1. Materials and Instruments
Doxorubicin hydrochloride and Poly-L-glutamic acid
(PLGA) were purchased from Sigma Company (USA).
Peptide c(RGDyK) was provided by ChuTai bio-
company (Shanghai, China). N-(3-Dimethylaminopropyl)-
N’-ethylcarbodiimide hydrochloride (EDC) was purchased
from Sigma Company (USA). U87MG cell was obtained
from the Type Culture Collection of the Chinese Academy
of Sciences (Shanghai, China). Follow reagents were
used in the cell culture and MTT experiment: RMPI1640
RMPI1640 was purchased from Gibco Company (Grand
Island, NY). Fetal bovine serum was purchased from
Sijiqing Company (Hangzhou, China). Thiazolyl blue
(MTT) and Dimethyl sulfoxide (DMSO) were obtained
from Amresco Company (Boise, USA). All other chemi-
cals were analytical grade.
2.4. Preparation of R-DMP
10 mg DMP was suspended in 10 ml PBS solution and
incubated with 5 mg 1-ethyl-3-[3-dimethylaminopropyl]
carbodiimide hydrochloride (EDC) and 5 mg N-hydroxy-
succinimide (NHS). After washing one time by PBS, 5 mg
PLGA was added for reaction with DMP through impulse
sonication for 2 h (each time ultrasonic bathing lasted
1 min with an interval of 5 min) at room temperature.
The PLGA modified DMP was collected by a Nd–Fe–
B magnet, and the supernatant solutions were removed.
Then 10 mL 0.1 M PBS solution was added to get rid of
the residual PLGA. After washing three times the parti-
cle was incubated with 5 mg EDC, 5mg NHS and DOX
solution (1 mg/mL) in 10 mL PBS solution by impulse
sonication for 3 h at room temperature. The product was
UV-visible spectrum was recorded on a Shimadzu
UV-3100 spectrophotometer (Kyoto, Japan). The ATR-IR
J. Nanosci. Nanotechnol. 14, 4858–4864, 2014
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