MRI-guided targeting delivery of doxorubicin with reduction-responsive lipid-polymer hybrid nanoparticles

Article abstract of DOI:10.2147/IJN.S143048

In recent years, there has been increasing interest in developing a multifunctional nanoscale platform for cancer monitoring and chemotherapy. However, there is still a big challenge for current clinic contrast agents to improve their poor tumor selectivity and response. Herein, we report a new kind of Gd complex and folate-coated redox-sensitive lipid-polymer hybrid nanoparticle (Gd-FLPNP) for tumor-targeted magnetic resonance imaging and therapy. Gd-FLPNPs can simultaneously accomplish diagnostic imaging, and specific targeting and controlled release of doxorubicin (DOX). They exhibit good monodispersity, excellent size stability, and a well-defined core-shell structure. Paramagnetic nanoparticles based on gadolinium-diethylenetriaminepentaacetic acid-biscetylamine have paramagnetic properties with an approximately two-fold enhancement in the longitudinal relaxivity compared to clinical used Magnevist. For targeted and reduction-sensitive drug delivery, Gd-FLPNPs released DOX faster and enhanced cell uptake in vitro, and exhibited better antitumor effect both in vitro and in vivo.

Full text of DOI:10.2147/IJN.S143048

International Journal of Nanomedicine
Dovepress
open access to scientific and medical research
Open access FullText article
O r I g I N a l r e s e a r c h
MrI-guided targeting delivery of doxorubicin
with reduction-responsive lipid-polymer hybrid
nanoparticles
This article was published in the following Dove Press journal:
International Journal of Nanomedicine
14 September 2017
Number of times this article has been viewed
Bo Wu1,2
Abstract: In recent years, there has been increasing interest in developing a multifunctional
nanoscale platform for cancer monitoring and chemotherapy. However, there is still a big chal-
lenge for current clinic contrast agents to improve their poor tumor selectivity and response.
Herein, we report a new kind of Gd complex and folate-coated redox-sensitive lipid-polymer
hybrid nanoparticle (Gd-FLPNP) for tumor-targeted magnetic resonance imaging and therapy.
Gd-FLPNPs can simultaneously accomplish diagnostic imaging, and specific targeting and
controlled release of doxorubicin (DOX). They exhibit good monodispersity, excellent
size stability, and a well-defined core-shell structure. Paramagnetic nanoparticles based on
gadolinium-diethylenetriaminepentaacetic acid-bis-cetylamine have paramagnetic proper-
ties with an approximately two-fold enhancement in the longitudinal relaxivity compared to
clinical used Magnevist. For targeted and reduction-sensitive drug delivery, Gd-FLPNPs
released DOX faster and enhanced cell uptake in vitro, and exhibited better antitumor effect
both in vitro and in vivo.
shu-Ting lu1
Kai Deng2
hui Yu2
can cui2
Yang Zhang2
Ming Wu2
ren-Xi Zhuo2
hai-Bo Xu1
shi-Wen huang2
1Department of radiology, Zhongnan
hospital of Wuhan University, 2Key
laboratory of Biomedical Polymers,
Ministry of education, Department of
chemistry,Wuhan University,Wuhan,
People’s republic of china
Keywords: redox-sensitive, tumor-targeted, gadolinium, contrast agents, PLGA
Introduction
Magnetic resonance imaging (MRI) has long been a powerful tool for efficient
diagnosis and therapeutic monitoring of cancers. Paramagnetic gadolinium ions
(Gd3+) with seven unpaired electrons can efficiently alter the relaxation time of the
surrounding water protons.1,2 As the major category of MRI contrast agents (CAs)
approved for clinical applications, Gd-based complexes such as gadodiamide, gado-
pentetate dimeglumine, gadoteridol, and gadoterate meglumine are widely used in
clinics. In recent years, impressive progress has also been made in the new form and
multifunction CA, including micelles,3,4 macromolecules,5 supramolecular aggregates,6
and liposomes,7 which have demonstrated good performance in enhancing the MR
contrast of early stage tumor. However, there is still a big challenge for current clinic
CAs to improve their poor tumor selectivity and response.
correspondence: shi-Wen huang
Key laboratory of Biomedical Polymers,
Ministry of education, Department
of chemistry, Wuhan University,
299 Bayi road, Wuhan 430072,
People’s republic of china
Tel/fax +86 27 6875 5317
Chemotherapy as the most effective treatment modality for cancer is still used as
the frontline approach in clinics. One major hindrance in chemotherapy is the systemic
distribution of anticancer drugs and concomitant serious side effects.8,9 As a solution to
this problem, many different effective response drug delivery systems were designed
to reduce severe side effects and improve the anticancer effect of anticancer drugs,10,11
such as redox-sensitive drug delivery platforms.12–15 Redox-sensitive nanocarriers
can achieve an annihilating effect and low toxicity on account of the sudden intracellular
email swhuang@whu.edu.cn
hai-Bo Xu
Department of radiology, Zhongnan
hospital of Wuhan University,
169 Donghu road, Wuhan 430071,
People’s republic of china
Tel/fax +86 27 6781 2533
email xuhaibo1120@hotmail.com
submit your manuscript | www.dovepress.com
Dovepress
International Journal of Nanomedicine 2017:12 6871–6882
6871
© 2017 Wu et al. This work is published and licensed by Dove Medical Press Limited. The full terms of this license are available at https://www.dovepress.com/terms.php
and incorporate the Creative Commons Attribution – Non Commercial (unported, v3.0) License (http://creativecommons.org/licenses/by-nc/3.0/). By accessing the work you
hereby accept the Terms. Non-commercial uses of the work are permitted without any further permission from Dove Medical Press Limited, provided the work is properly attributed. For permission
for commercial use of this work, please see paragraphs 4.2 and 5 of our Terms (https://www.dovepress.com/terms.php).
http://dx.doi.org/10.2147/IJN.S143048

Wu et al
Dovepress
burst of encapsulated drugs caused by the cleavage of dis-
ulfide bonds in the redox environment of cancer cells.16–18
Our earlier studies have demonstrated the advantages of the
amphiphilic reduction-sensitive polymer of monomethoxy-
poly-(ethylene glycol)-S-S-hexadecyl (mPEG-S-S-C16) for
the effective intracellular delivery of anticancer drugs.13,19
To further improve the therapeutic efficiency and reduce
side effects, a better strategy was to develop molecular-
targeted nanoparticle (NP) therapeutic carriers. Folate recep-
tor (FR), an attractive overexpressed receptor by several
human tumors such as ovarian cancer, breast cancer, and
epidermoid carcinoma of the oral cavity20–22 presented an
effective means for selective delivery of therapeutics. Folate,
which can bind very firmly to FRs (Kd∼10−10 M),23 has been
proved to effectively improve the drug uptake efficiency.
Hence, we developed a kind of folate-targeted, Gd-coated,
and redox-sensitive NP that can achieve both imaging and
therapy with redox-sensitivity and active targeting ability.
Lipid-polymer hybrid nanoparticles (LPNPs), which com-
bine the biosafety of liposomes and versatility of polymeric
NPs into a single delivery system, were a good choice for
this multifunctional nanocarrier, which uses folate as the
targeted ligand and amphiphilic reduction-sensitive poly-
mer to achieve intracellular release, and then improve the
therapeutic effect. Compared to traditional nanoplatforms,
$
%
3/*$ꢀSRO\PHUꢀFRUH
/HFLWKLQ
3(*ꢁNꢂ6ꢂ6ꢂ&ꢃꢄ
ꢀ'63(ꢂ3(*ꢁNꢂIRODWH
*Gꢂ'73$ꢂ%&ꢃꢄ
'2;
*6+
'HDGꢀFHOO
Figure 1 schematic illustration of the formation of gd-FlPNPs and cancer-targeted
intracellular drug delivery.
Notes: (A) The amphiphilic mPeg-s-s-c16, contrast agent gd-DTPa-Bc16,
targeted ligand, and anticancer drug DOX were self-assembled into gd-FlPNPs.
(B) Uptake of gd-FlPNPs by cancer cells via the active targeting strategy and the
anticancer drug was rapidly released for cancer therapy upon the triggering of gsh
in cancer cells.
Abbreviations: gd-FlPNPs, gd-coated folate-targeted lipid-polymer hybrid
nanoparticles; DOX, doxorubicin; gsh, glutathione; mPeg-s-s-c16, monomethoxy-
poly-(ethylene glycol)-s-s-hexadecyl; gd-DTPa-Bc16, gadolinium-diethylene-
triaminepentaacetic acid-bis-tetradecylamide; Plga, poly(d,l-lactide-co-glycolide);
DsPe-Peg2k-folate,
(polyethylene glycol) 2000-folate.
1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-carboxy
Gd-coated folate-targeted lipid-polymer hybrid nanopar- were also prepared using the same above-mentioned method.
ticles (Gd-FLPNPs) can simultaneously accomplish specific DSPE-PEG2k and DSPE-PEG2k-folate were purchased
targeting, controlled release of anticancer drugs, MRI, and from Shanghai Advanced Vehicle Technology Co, Ltd
simultaneous therapy. The Gd-FLPNPs were composed of (Shanghai, People’s Republic of China). mPEG2k-S-S-C16
a biodegradable hydrophobic poly(d,l-lactide-co-glycolide) was synthesized using a method reported in our previous
(PLGA) core and a paramagnetic liposome shell which was studies.14 The consumables of cell culture in this work were
based on gadolinium-diethylenetriaminepentaacetic acid- purchased from Beijing Dingguo Changsheng Biotechnology
bis-tetradecylamide (Gd-DTPA-BC16), 1,2-distearoyl-sn- Co, Ltd (Beijing, People’s Republic of China). PLGA
glycero-3-phosphoethanolamine-N-carboxy (polyethylene (75:25, molecular weight: 90,000–126,000) and lecithin
glycol) 2000-folate (DSPE-PEG2k-folate), and mPEG-S- were purchased from Sigma-Aldrich (St Louis, MO, USA).
S-C16. The PLGA core was employed to load and release Diethylenetriaminepentaacetic acid (DTPA), N,N-dimethyl
hydrophobic drugs. The paramagnetic liposome shell offers formamide (DMF), DL-dithiothreitol (DTT), triethylamine
many advantages such as folate targeting provided by DSPE- (TEA), and dimethylsulfoxide (DMSO) were all purchased
PEG2k-folate, redox-sensitivity provided by mPEG-S-S-C16, from Shanghai Chemical Co (Shanghai, People’s Republic
and contrast-enhanced ability provided by Gd-DTPA-BC16. of China). All solvents used in this study were of high-
The structure of Gd-FLPNPs is shown in Figure 1.
performance liquid chromatography grade.
Materials and methods
Materials
synthesis of ligands
In brief, DTPA (3.92 g, 10 mmol) was dissolved in acetic
The synthesis of DTPA-BC16 and Gd-DTPA-BC16 was anhydride (19 mL, 200 mmol) at 70°C for 10 hours under
carried out by the method of Kimpe et al.24 Gd-FLPNPs argon atmosphere. The solvent was evaporated under reduced
were prepared using a previously reported single-step pressure. The obtained crude product of DTPA dianhydride
assembly method.14 For comparison, non-targeted Gd-LPNPs was washed twice with acetic anhydride. Then, DTPA
submit your manuscript | www.dovepress.com
International Journal of Nanomedicine 2017:12
6872
Dovepress

Dovepress
MrI-guided targeting delivery of doxorubicin
dianhydride (714 mg, 2 mmol) and cetylamine (968 mg, Brookhaven Instruments Co, Holtsville, NY, USA). Trans-
4 mmol) were dissolved in anhydrous DMF for 12 hours. mission electron microscopy (TEM, JEM-100CX; JEOL,
After solvent evaporation under reduced pressure, the Tokyo, Japan) was used to observe the micelle morphology
obtained crude product was purified by preparative thin layer at an operating voltage of 100 kV. Each result was an average
chromatography on silica.
of triplicate measurements. X-ray photoelectron spectros-
copy spectra (XPS, Kratos XSAM800 spectrometer; Kratos,
Manchester, UK) was employed to investigate the surface
chemistry of the NPs, using Mg Kα radiation (1,253.6 eV)
synthesis of the gd-DTPa-Bc16
complexes
Gd-DTPA-BC16 was synthesized by the general procedure as the exciting source operated at 11.5 kV and 17 mA.
as follows: hydrated GdCl3 salt (1.1 mmol) in H2O (1 mL)
Drug encapsulation efficiency (EE)
was added to the ligand (Gd-DTPA-BC16, 1 mmol) dissolved
in pyridine (30 mL), and the mixture was heated at 70°C and responsive drug release
for 3 hours. The solvents were evaporated under reduced To evaluate the drug EE and loading efficiency (LE), a
pressure and the crude product was then heated at reflux in predetermined aliquot of Gd-FLPNPs or Gd-LPNPs was
ethanol for 1 hour. After the mixture had cooled to room collected by freeze drying, and then the dry NPs were dis-
temperature, the complex was filtered off and dried in vacuo. solved in DMSO. After that, the DOX concentration in
The absence of free gadolinium was checked with xylenol DMSO was measured by fluorescence measurement (excita-
orange indicator.
tion at 485 nm) using a calibration curve constructed from
DOX solutions with different DOX concentrations. EE was
calculated as (actual amount of drug encapsulated in NPs)/
Preparation of paramagnetic gd-FlPNPs
Gd-FLPNPs were prepared using a previously reported (initial amount of drug used in the fabrication of NPs) ×100%.
single-step assembly method. In brief, prior to the Gd- LE (%) = (amount of drug in particles/amount of the feeding
FLPNPs preparation, doxorubicin hydrochlorate (DOX⋅HCl) material and drug) ×100%.
was stirred with twice the molar amount of TEA in DMF for
Two milliliters (DOX, 1 mg/mL) of Gd-FLPNPs were
10 hours to obtain lipophilic doxorubicin (DOX). DOX transferred to a dialysis tube (MWCO 8,000). Then, it was
(5 mg) was dissolved in 10 mL of DMF. Then the polymer of immersed into a tube containing 10 mL of phosphate-buffered
PLGA (40 mg) was added to the solution and stirred at room saline (PBS; 10 mM, pH 7.4) with or without 10 mM DTT
temperature for 2 hours. DSPE-PEG2k-folate, mPEG-S-S-C16, in a shaker shaking at 120 rpm and 37°C. At designated time
Gd-DTPA-BC16, and lipid lecithin (weight ratio 1/6/6/2, total intervals, 5 mL of the external buffer was replaced with the
weight 30 mg) were dissolved in 30 mL of 4 wt % ethanol corresponding fresh buffer solution. Then, DOX quantity
aqueous solution at 65°C, and the DOX/PLGA solution was determined by fluorescence measurement (excitation
was added dropwise under gentle stirring. Then, the mixed at 485 nm) using a calibration curve constructed from DOX
solution was vortexed vigorously for 3 minutes followed solutions with different DOX concentrations. The error bars
by dialyzing against ultrapure water at 25°C for 48 hours. were obtained from triplicate samples.
Finally, the NPs were concentrated using an Amicon
Ultra-4 Centrifugal Filter (EMD Millipore, Billerica, MA, cell culture
USA; molecular weight cut-off [MWCO] =8,000). To Cervical cancer cell (KB cell) was purchased from the China
serve as a control, non-targeted paramagnetic Gd-LPNPs Center for Type Culture Collection (Wuhan University)
and FLPNPs were also prepared by the above process, and cultured in RPMI 1640 medium, folate-free RPMI
and DSPE-PEG2k-folate or Gd-DTPA-BC16 was replaced 1640 medium, supplemented with 4×10−3 M L-glutamine,
by DSPE-PEG2k. Additionally, DOX-free Gd-FLPNPs or 10% fetal bovine serum (FBS), and 1% antibiotics at 37°C
DOX-free Gd-LPNPs were also prepared using the above in a humidified atmosphere containing 5% CO2.
protocol but without DOX.
animals and tumor-bearing mice models
Female BALB/c nude mice were obtained from Beijing HFK
Physicochemical characterizations
of gd-FlPNPs and gd-lPNPs
Bioscience Co Ltd (Beijing, People’s Republic of China)
The particle size and size distribution of the drug loaded NPs with body weights of 18–20 g and housed under standard
were measured by dynamic light scattering (DLS, 90Plus, pathogen-free conditions (60% relative humidity, 20°C
submit your manuscript | www.dovepress.com
International Journal of Nanomedicine 2017:12
6873
Dovepress

Wu et al
Dovepress
room temperature) with a 12 hours light/12 hours dark cycle. in 4 mL of folic acid deficient 1640 medium containing 10%
Animals were acclimated to this condition for 5 days prior to FBS, and then cultured with Gd-FLPNPs or Gd-LPNPs at
treatment. These nude mice were subcutaneously inoculated a final DOX concentration of 2 μg/mL. At predetermined
with KB cells (2×106 cells per mice) at backside to establish intervals, the cells were washed with PBS to remove the
the subcutaneous transplanted tumor model. Procedures free NPs that did not enter the cells prior to fluorescence
involving animals and their care were conducted in confor- observation.
mity with EU Directive 2010/63/EU for animal experiments
In vitro cellular uptake by confocal laser
scanning microscopy (clsM)
CLSM (Nikon TE2000; Nikon Corporation, Tokyo, Japan)
and was approved by the Animal Welfare Committee of the
Animal Experiment Center of Wuhan University.
In vitro MrI property
was used to examine the intracellular distribution of
Gd-FLPNPs, Gd-LPNPs, and Magnevist were diluted with DOX. KB cells were seeded on slides at a density of 5.0×104
deionized water to obtain various concentrations of CA. cells/well in 1 mL of folic acid deficient 1640 medium
Two hundred microliters of different concentrations of containing 10% FBS. The cells were then incubated with
samples were added to a 96-well plate. The in vitro MRI Gd-FLPNPs or Gd-LPNPs at a final DOX concentration of
test was performed with a Siemens Prima 3.0T MRI scanner 2 μg/mL. At predetermined intervals, the cells were washed
(Siemens, Munich, Germany). T1-weighted MR images of with PBS and fixed with 4% (w/v) paraformaldehyde aqueous
Gd-FLPNPs, Gd-LPNPs, and a commercially available CA solution for 10 minutes. The slides were then stained with
(Magnevist) were obtained under the following experimental Hoechst 33258 (5 mg/mL in PBS) at 37°C for 10 minutes.
conditions: recycle time 700 ms, echo delay time 12 ms. For The fixed cell monolayer was finally observed by CLSM.
relaxivity-coefficient measurements, NP samples were pre-
pared with varying Gd3+ concentrations. The mean value of endocytosis inhibition
a region of interest was used to determine the relaxivity (r1). KB cells were seeded on slides on a 6-well plate at a density of
r1 which reflected the proton-relaxation-enhancement ability 5.0×104 cells/well in 1 mL of folic acid deficient 1640 medium
of NPs in water was calculated as follows: r1= (1/T1 − 1/T1d)/ containing 10% FBS. Then, the cells were cultured for 1 day
[Gd], where 1/T1 was the longitudinal relaxation rate con- at 37°C in 5% CO2 atmosphere. The cells were pretreated for
trast with the presence of a paramagnetic species, 1/T1d 30 minutes with three different endocytosis inhibitors sepa-
represented longitudinal relaxation rate contrast with the rately at concentrations of 5 mM methyl-beta-cyclodextrin,
absence of paramagnetic species, and [Gd] represented the 0.45 mM sucrose, or 5 mM cytochalasin D. Then, DOX-
concentration of paramagnetic NPs (mM).
loaded Gd-FLPNPs were added at a final DOX concentration
of 2 μg/mL, followed by incubation for 2 hours. The subse-
quent steps were similar to those described in the “In vivo
In vivoT1-weighted MrI
To evaluate the enhancement of Gd-FLPNPs in MRI, C6 T1-weighted MRI” section. The group with test materials but
cells in the tumor-bearing mouse were scanned by a Siemens without inhibitor treatment was used as a control.
Prima 3.0T MRI scanner. Before and after intravenous (iv)
injection of Gd-FLPNPs, the images including T1-WI and In vitro cytotoxicity assay
its pseudocolor images of the nude mouse were obtained. Cytotoxicity of Gd-FLPNPs or Gd-LPNPs was evaluated by
During the whole scan process, the mouse was anesthetized 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium
by isoflurane and maintained at normal temperature. The bromide (MTT) assay. Briefly, KB cells were seeded on
T1-weighted image parameters were as follows: repetition a 96-well plate (Costar, Cambridge, MA, USA) at a den-
time 700 ms; echo time 12 ms; field of view 120×120 mm; sity of 5.0×103 cells/well in 100 μL of folic acid deficient
matrix size 400×400; slice thickness 2.0 mm; number of 1640 medium containing 10% FBS. The cells were cultured
acquisitions 14.
for 24 hours at 37°C in 5% CO2 atmosphere. Afterwards, the
nonloaded nanocarrier, DOX-free Gd-FLPNPs, DOX-free
Gd-LPNPs, DOX-loaded Gd-FLPNPs, DOX-loaded Gd-
LPNPs, and free DOX at various concentrations, were added
Evaluation of cellular uptake by flow
cytometry (FcM)
The cellular uptake of the DOX-loaded NPs was confirmed to each well. After incubation for 8 hours, the media were
by fluorescence microscopy (Epics XL). KB cells were replaced by fresh folic acid deficient 1640 medium. After
incubated in 6-well plates at a density of 4.0×105 cells/well culture for 38 hours, MTT solution (5 mg/mL in PBS, 20 μL)
submit your manuscript | www.dovepress.com
International Journal of Nanomedicine 2017:12
6874
Dovepress

Dovepress
MrI-guided targeting delivery of doxorubicin
was added to each well and incubated for 4 hours. The media Then, Gd3+ was incorporated into DTPA-BC16. The structures
were removed and 150 μL of DMSO was added to each well
to dissolve the formazan blue crystal. The absorbance of the
solution was measured using a microplate reader at 570 nm.
Cell viability was calculated as a percentage of the control
(cells receiving no treatment).
of Gd-DTPA-BC16 and mPEG-S-S-C16 were identified by
1H NMR spectra as shown in Figures 2B and S2, and by
infrared spectroscopy (Figure 2C). After that, Gd-FLPNPs
were prepared using a reported self-assembly method.25
Gd-FLPNPs were made up of two components (Figure 1):
a hydrophobic PLGA core, and a paramagnetic and redox-
sensitive liposome shell. The elements on the core-shell
Gd-FLPNPs surface were identified according to the specific
binding energy of Gd4d using XPS (Figure 3D and E), which
indicated the successful coating of Gd-DTPA-BC16 on the
NPs. As shown in Figure 3A, a clear core-shell structure is
visible from the TEM images. The images also show that
Gd-FLPNPs were dispersed individually with a well-defined
spherical shape and a smooth surface. The paramagnetic
lipid shell about 10–20 nm was fused onto the hydrophobic
PLGA core (Figure 3A). The merit of this structure is that
poorly soluble drugs can be encapsulated into the hydro-
phobic PLGA cavity with a high LE. At the same time, the
multifunctional liposome shell offers many advantages such
as folate targeting, redox-sensitivity, and contrast-enhanced
antitumor assessment and histological
analysis in vivo
The sizes of the tumor in the mice models were measured
every other day. When the tumor grew up to ∼50 cm3,
20 mice were divided into four groups and intravenously
injected with PBS, free DOX, Gd-LPNPs, and Gd-FLPNPs.
Tumor volume was estimated by the following equation:
V = a×b2/2, where a and b were the longest and shortest
diameters, respectively.
Results
Preparation of gd-FlPNPs
The synthesis process of Gd-DTPA-BC16 and mPEG-S-S-C16
are presented in Figures 2A and S1. The carboxyl groups of
DTPA were combined with the amino groups of cetylamine. ability for diagnosis and therapy.
$
+22&
%
E
F
D
D
D
D
E
F
+22&
&22+
2
2
2
1
1
1
&22+
&22+
+ꢀ&
+ꢀ&
3\ULGLQH
&
+
ꢀꢀ+1
&
2
&
2
1+&ꢃꢄ+
ꢀꢀ
ꢃꢄ
E
1
1
1
ꢎ
ꢊ
&22+
JIHG
GHIJ
+22&
&22+
I
&'ꢀ2'
2
2
2
2
2
2
1
1
1
&22+
&+&,ꢀ
'0)
ꢁꢂ&ꢃꢄ+ꢀꢀ1+ꢁ
F
G
J
H
E
+22&
&22+
'73$ꢀ%&ꢁꢂ
D
1
1
1
&
+
ꢀꢀ+1
&
2
&
2
1+&ꢃꢄ+
ꢀꢀ
ꢃꢄ
&22+
*G&,ꢀ
ꢅꢆꢇ ꢈꢆꢇ ꢉꢆꢇ ꢄꢆꢇ ꢇꢆꢇ ꢊꢆꢇ ꢀꢆꢇ ꢁꢆꢇ ꢃꢆꢇ ꢋꢆꢇ ±ꢋꢆꢇ
SSP
&
'73$ꢌ%&ꢃꢄ
+22&
&22+
1
1
1
&
+ꢀꢀ+1
&
2
&
2
1+&ꢃꢄ+
ꢀꢀ
ꢃꢄ
&22+
*Gꢀ'73$ꢀ%&ꢁꢂ
*Gꢌ'73$ꢌ%&ꢃꢄ
ꢀꢍꢀꢇꢉ
*Gꢀꢎ
ꢃꢍꢀꢅꢈ
ꢃꢍꢋꢋꢋ
ꢃꢍꢄꢁꢇ
ꢀꢍꢋꢋꢋ
ꢁꢍꢋꢋꢋ
ꢋ
:DYHQXPEHUꢃꢄFP±ꢁꢅ
Figure 2 (A) synthesis of DTPa-Bc16 and gd-DTPa-Bc16. (B) 1h NMr spectra of DTPa-Bc16 polymer in cD3OD and cDcl3 (ppm). (C) Infrared spectroscopy of
gd-DTPa-Bc16 and DTPa-Bc16.
Abbreviations: DMP, N,N-dimethylformamide; gd-DTPa-Bc16, gadolinium-diethylenetriaminepentaacetic acid-bis-tetradecylamide; NMr, nuclear magnetic resonance.
submit your manuscript | www.dovepress.com
International Journal of Nanomedicine 2017:12
6875
Dovepress

Wu et al
Dovepress
$
%
&
*Gꢃ)/313VꢂLQꢂ3%6
*Gꢃ)/313VꢂLQꢂꢀꢁꢂP0ꢂ'77
ꢆꢁ
ꢀꢅ
ꢀꢁ
ꢅ
&RUH
'77
6KHOO
ꢀꢁꢁꢂQP
ꢀꢁꢁꢂQP
ꢁ
ꢀꢁꢁ
ꢀꢄꢁꢁꢁ
ꢀꢁꢄꢁꢁꢁ
6L]HꢀꢁQPꢃ
ꢀꢁꢁ
ꢉꢁ
ꢈꢁ
ꢇꢁ
ꢆꢁ
ꢁ
*GꢇG
'
(
)
*Gꢃ)/313V
)/313V
*GꢇG
*Gꢃ)/313VꢂZLWKꢂꢀꢁꢂP0ꢂ'77
*Gꢃ)/313VꢂZLWKRXWꢂ'77
ꢁ
ꢆꢁꢁ
ꢇꢁꢁ
ꢈꢁꢁ
ꢉꢁꢁ ꢀꢁꢁ
ꢀꢆꢁ
ꢀꢇꢁ
ꢀꢈꢁ
ꢀꢉꢁ
ꢆꢁꢁ
ꢁ
ꢀꢁ
ꢆꢁ
ꢊꢁ
ꢇꢁ
ꢅꢁ
%LQGLQJꢀHQHUJ\ꢀꢁH9ꢃ
%LQGLQJꢀHQHUJ\ꢀꢁH9ꢃ
5HOHDVHꢀWLPHꢀꢁKꢃ
Figure 3 characterizations of gd-FlPNPs.
Notes: (A) TeM image of gd-FlPNPs. (B) TeM image of gd-FlPNPs treated with DTT (10 mM) for 4 hours. (C) size change of gd-FlPNPs in response to 10 mM DTT in
PBs after 4 hours (0.01 M, ph =7.4) determined by Dls. (D) XPs spectra of the samples. (E) gd4d signal spectra of gd-FlPNPs. (F) redox-triggered release of DOX from
gd-FlPNPs in PBs (0.01 M, ph 7.4) with or without 10 mM DTT.
Abbreviations: gd-FlPNPs, gd-coated folate-targeted lipid-polymer hybrid nanoparticles; DOX, doxorubicin; TeM, transmission electron microscopy; DTT, Dl-
dithiothreitol; PBs, phosphate-buffered saline; Dls, dynamic light scattering; XPs, X-ray photoelectron spectroscopy.
of size distribution of Gd-FLPNPs in response to 10 mM DTT
characterization of gd-FlPNPs
was monitored by TEM (Figure 3B) and DLS (Figure 3C).
Due to the cleavage of the disulfide linkage, the size of Gd-
FLPNPs significantly increased after 4-hour co-incubation
of DTT, indicating the falling off of the hydrophilic PEG
shell from the Gd-FLPNPs. Destabilization and aggrega-
tion of the hydrophobic inner core in the reducing environ-
ments are conducive to the release of drug and CAs in the
tumor tissue, and result in tumor-specific enhanced imaging
and therapy.
The size and size distribution of Gd-FLPNPs were charac-
terized by DLS (Table 1; Figure 3C). The average size of
Gd-FLPNPs was about 120–130 nm, which is conducive to
satisfactory drug accumulation in the tumor site through the
enhanced permeability and retention (EPR) effect.26,27 The
polydispersity of Gd-FLPNPs was 0.106, which indicated
a unimodal size distribution. In this study, DOX was suc-
cessfully loaded into Gd-FLPNPs with a satisfying loading
efficiency (LE) and encapsulation efficiency (EE); the LE
and EE of Gd-FLPNPs were about 5.4% and 76.3%, respec-
tively (Table 1).
In vitro redox-sensitivity release
The in vitro redox-sensitive release behavior of Gd-FLPNPs
was further analyzed using a dialysis method. As shown
in Figure 3, the Gd-FLPNPs incubated with DTT showed
more efficient and fast release of DOX from Gd-FLPNPs
in the same period. In contrast, less DOX was released in
the absence of DTT. For Gd-FLPNPs, about 79.3% of the
payload was released over a period of 24 hours in the presence
of 10 mM DTT, while 63.4% of the payload was released in
the absence of DTT. This result may be related to the cleav-
age of the disulfide bond of mPEG-S-S-C16 in the reducing
environment, which resulted in the dissociation of the NPs
structure. These results suggested that the Gd-FLPNPs could
As a redox-sensitive NP, Gd-FLPNPs exhibit a rapid
response to reducing environments (Figure 3B). The change
Table 1 Physicochemical characterization of gd-FlPNPs and
gd-lPNPs
Sample
Particle
PDI
EE (%)
LE (%)
size (nm)
gd-FlPNPs
gd-lPNPs
122.6±5.2
127.3±4.7
0.106
0.154
76.3
74.2
5.4
5.3
Note: Data represent mean ± sD, n=3.
Abbreviations: gd-FlPNPs, gd-coated folate-targeted lipid-polymer hybrid
nanoparticles; gd-lPNPs, gd-coated lipid-polymer hybrid nanoparticles; sD, standard
deviation; PDI, polydispersity index; EE, encapsulation efficiency; LE, loading efficiency.
submit your manuscript | www.dovepress.com
International Journal of Nanomedicine 2017:12
6876
Dovepress

Dovepress
MrI-guided targeting delivery of doxorubicin
release loading responsively, and may be a potential intracel- Gd-LPNPs. This result suggested that the folate on the surface
lular environment-sensitive drug delivery system.
of NPs facilitated the entry of Gd-FLPNPs into cells, which
is consistent with FCM results. These results fully proved
that Gd-FLPNPs were transported to cells through an FRs-
mediated endocytosis process.
Targeted cell uptake and profile
The targeted cellular uptake profiles of Gd-FLPNPs were
examined with folate overexpressing KB cells (human
oral epidermoid carcinoma cell line) using FCM. With the
absence of folate in the culture medium, the KB cells with
Gd-FLPNPs resulted in stronger DOX fluorescence than with
Gd-LPNPs, in both the cytoplasm and nuclei. The mean DOX
fluorescence value was 28.2 after 4 hours of incubation with
Gd-FLPNPs in a folate-free medium, while it was 19.6 after
incubation with non-targeted lipid-polymer hybrid nanopar-
ticles (Gd-LPNPs) (Figure 4). We also examined the effects
of the addition of folate into the medium. When folate-free
medium was replaced with folate-contained medium, there
was little difference in the cellular uptake of DOX between
Gd-FLPNPs and Gd-LPNPs. As a result of the competitive
To further study the targeting efficacy of Gd-FLPNPs, KB
cells were incubated with folate-targeted and non-targeted
paramagnetic NPs. Figure 6C shows the Gd concentration,
which was measured from the lysate of KB cells incubated
with Magnevist, non-targeted Gd-LPNPs, and folate-targeted
Gd-FLPNPs. Clearly, Gd3+ concentrations were higher after
incubation with both types of NPs than with Magnevist.
In addition, the uptake of Gd-FLPNPs (0.24 mg/L) was
much more efficient than Gd-LPNPs (0.15 mg/L) by KB cells
with an FRs-mediated endocytosis process. This means that
Gd-LPNPs could provide high delivery efficiency of CAs
and acquire higher contrast MRI.
binding to FRs, the folate in the culture medium prevented In vitro MrI
Gd-FLPNPs from transporting into KB cells. The internaliza- Proton longitudinal relaxivity (r1), which is an important
tion pathway of Gd-LPNPs was also evaluated using three parameter revealing the ability of a CA for MRI images, was
types of endocytosis inhibitors. As shown in Figure 4B, the calculated from the change of water proton relaxation rate
mean DOX fluorescence intensity decreased to 43% after 1/T1 (s−1) per mM concentration. To obtain the T1-weighted
treatment with hypertonic sucrose, which indicated that the MRI images and r1, solutions containing different
pathway of Gd-FLPNPs into cells depended on clathrin- concentrations of Gd-FLPNPs or Magnevist were placed
mediated endocytosis.28
in a 96-well plate and tested by a Siemens Prima 3.0T MRI
Cellular uptakes of Gd-FLPNPs and Gd-LPNPs by KB scanner. T1-weighted MRI images of the phantom are shown
cells were further studied by CLSM. The results show that in Figure 6A. The signal intensity of Gd-FLPNPs was clearly
Gd-FLPNPs produced stronger DOX fluorescence than better than that of Magnevist. After fitting, the r1 of Gd-
Gd-LPNPs after 4 hours of incubation (Figure 5). More- FLPNPs was 8.02 mM−1 s−1 as shown in Figure 6B. In contrast,
over, when KB cells were incubated in the folate medium, the r1 value of small molecule Magnevist was 4.61 mM−1 s−1.
Gd-FLPNPs showed a similar extent of cellular uptake as The results indicate that Gd-FLPNPs hold better contrast
$
%
ꢃꢁ
ꢀꢂ
ꢀꢋ
ꢀꢅ
ꢀꢃ
ꢀꢁ
ꢂ
ꢋ
ꢅ
ꢃ
ꢁ
ꢅꢂꢉꢊ
ꢆꢁ
ꢅꢁ
ꢄꢁ
ꢃꢁ
ꢀꢁ
ꢁ
ꢀꢊꢉꢆ
ꢀꢋꢉꢆ
ꢂꢁ
ꢂꢁ
ꢀꢆꢉꢀ
ꢄꢅꢉꢆ
ꢄꢄꢉꢄ
ꢊꢉꢆ
ꢁꢉꢂ
ꢁꢉꢂ
ꢁ
ꢀꢁꢁ
ꢁ
ꢀꢁꢁ
ꢀꢁꢀ
ꢀꢁꢃ
ꢀꢁꢄ
ꢀꢁꢅ
ꢀꢁꢀ
ꢀꢁꢃ
ꢀꢁꢄ
ꢀꢁꢅ
Figure 4 study of targeted cell uptake and internalization pathway by FcM.
Notes: (A) Quantitative flow cytometry analysis of the cellular DOX red fluorescence MFI values of KB cells incubated with Gd-FLPNPs for 10 hours; DOX dosage was
2 μg/ml. (B) Effect of endocytosis inhibitors on the uptake of Gd-FLPNPs in KB cells using flow cytometry analyses; DOX dosage was 2 μg/ml.
Abbreviations: FCM, flow cytometry; Gd-FLPNPs, Gd-coated folate-targeted lipid-polymer hybrid nanoparticles; DOX, doxorubicin; Gd-LPNPs, Gd-coated lipid-polymer
hybrid nanoparticles; MBCD, methyl-beta-cyclodextrin; MFI, mean fluorescence intensity of cellular DOX red fluorescence.
submit your manuscript | www.dovepress.com
International Journal of Nanomedicine 2017:12
6877
Dovepress

Wu et al
Dovepress
'2;
+RHFKVW
0HUJHG
$
%
&
Figure 5 clsM images of KB cells incubated with (A) gd-FlPNPs, (B) gd-lPNPs, and (C) gd-FlPNPs pretreated with excess of folate acid for 4 hours.
Note: DOX: red fluorescence images; nuclei: blue fluorescence images; blue and red fluorescence: merged images (scale bar, 20 μm).
Abbreviations: gd-FlPNPs, gd-coated folate-targeted lipid-polymer hybrid nanoparticles; DOX, doxorubicin; gd-lPNPs, gd-coated lipid-polymer hybrid nanoparticles;
clsM, confocal laser scanning microscopy.
capability than Magnevist at the same concentration. As T1-weighted MR image reached a maximum 4 hours after
reported previously, the proton relaxivity of lanthanide ions injection. Then, the signal intensity decreased gradually with
in water could be influenced by a combination of factors, the time prolonging. Further quantitative analysis showed
that may be the reason of improvement of macromolecular that MR signals revealed T1 signal intensity enhancement
CAs.29,30 It has been demonstrated that due to a prolongation by 36%, suggesting high tumor accumulation of the NPs
of the rotational correlation time (τR), relaxivity improves after systemic administration. Additionally, due to the EPR
with the incorporation of paramagnetic ions into a macro- effect of NPs, Gd-FLPNPs provided a long time window
molecular structure.31,32
to obtain high-quality MRI for guidance and to moni-
tor the effect of chemotherapy compared to commercial
CA (Magnevist).
In vivo MrI
To study the MRI contrast effect of Gd-FLPNPs,
T1-weighted MR images were acquired using nude mice
antitumor effects of gd-FlPNPs in vitro
bearing C6 tumor xenografts after injection of Gd-FLPNPs and in vivo
at different time points. Compared with the image of The antitumor effects of Gd-FLPNPs, Gd-LPNPs, and
preinjection, an obvious enhanced effect was found in free DOX against KB cells were evaluated by the MTT
T1-weighted MR image of the tumor as shown in Figure 6D. assay. Before that, the KB cells were incubated with dif-
Obvious T1 hyperintense signals could be found after 1 hour ferent concentrations of DOX-free Gd-FLPNPs, DOX-free
injection of Gd-FLPNPs, and the contrast enhancement in Gd-LPNPs, or Magnevist. The result proves that neither the
submit your manuscript | www.dovepress.com
International Journal of Nanomedicine 2017:12
6878
Dovepress

Dovepress
MrI-guided targeting delivery of doxorubicin
ꢀꢀꢀ
&
$
%
ꢉꢆꢀ
ꢅꢆꢇ
ꢅꢆꢀ
ꢂꢆꢇ
ꢂꢆꢀ
ꢀꢆꢇ
ꢀꢆꢀ
*Gꢈ)/313V
0DJQHYLVW
ꢀꢆꢅꢃ
ꢀꢆꢅꢇ
ꢀꢆꢅꢀ
ꢀꢆꢂꢇ
ꢀꢆꢂꢀ
ꢀꢆꢀꢇ
ꢀꢆꢀꢀ
ꢀꢆꢉꢀ
ꢀꢆꢂꢇ
ꢀꢆꢂꢇ
Uꢂ ꢄꢆꢀꢅ
ꢀꢆꢀꢊꢇꢇ
ꢀꢆꢀꢉꢊꢇ
Uꢂ ꢃꢆꢋꢂ
ꢀꢆꢀꢇ
ꢀꢆꢀ
ꢀꢆꢂ
ꢀꢆꢅ
ꢀꢆꢉ
*GꢀFRQFHQWUDWLRQꢀꢁP0ꢂ
'
ꢀꢁK
ꢂꢁK
ꢃꢁK
ꢄꢁK
ꢅꢃꢁK
Figure 6 The contrast effect of gd-FlPNPs.
Notes: (A) Phantom Mr images of gd-FlPNPs in comparison to Magnevist showing T1-weighted bright contrast. (B) relaxivity plot for gd-FlPNPs and Magnevist. (C) IcP-
aes measurements of cellular gd concentration with 10 hours post-uptake of gd-FlPNPs ***(P,0.001). (D) In vivo Mr images of human oral epidermoid carcinoma-bearing
mice pre- and post-intravenous injection of gd-FlPNPs. The red circled area marks the tumor location.
Abbreviations: Mr, magnetic resonance; gd-FlPNPs, gd-coated folate-targeted lipid-polymer hybrid nanoparticles; gd-lPNPs, gd-coated lipid-polymer hybrid
nanoparticles; IcP-aes, inductively coupled plasma atomic emission spectroscopy; DTPa, diethylenetriaminepentaacetic acid.
NPs nor Magnevist has a negligible effect on the survival PBS solution of Gd-FLPNPs and Gd-LPNPs. As shown
rate of KB cells at any concentration (Figure 7A). As shown in Figure 7C and D, the mice inoculated tumors with PBS
in Figure 7B, all DOX-loaded materials notably inhibited the only grew rapidly. On day 14, the mean tumor volume was
growth of KB cells. The inhibitory effects on the cell viability about nine times as big as the initial volume. Free DOX
were dose-dependent. The IC50 of Gd-FLPNPs, Gd-LPNPs, group showed antitumor effect to some extent against the
and free DOX were about 0.98, 1.75, and 0.34 μg/mL, respec- subcutaneous tumor in mice, especially in the early stage
tively. Since small molecules can be more easily transported of tumor, and then the tumor continued to increase in size.
into cells and nuclei by passive diffusion, free DOX was more In contrast, after treating with Gd-FLPNPs, tumor growth
cytotoxic to KB cells than the NPs as shown in Figure 7B. was inhibited by 64%, while in the group of Gd-LPNPs it
Predictably, at the same dose of DOX, the cytotoxicity of was inhibited by 45%. Compared with the non-targeted
Gd-FLPNPs was significantly higher than that of Gd-LPNPs, group, the folate active targeting ligand enables sufficient
which was consistent with the results observed by CLSM and accumulation of Gd-FLPNPs in tumor tissues, and then
FCM. Moreover, with 2 mM folate in the medium, the cyto- showed significant antitumor effects. The above results
toxicity of Gd-FLPNPs was noticeably reduced and became confirmed the potential folate-targeted antitumor effect of
nearly equivalent to that of Gd-LPNPs. This fact indicated Gd-FLPNPs.
that the cell uptake of Gd-FLPNPs was an FRs-mediated
endocytosis process.
The potential in vivo toxicity induced by different treat-
ments was evaluated by the body weight of animals after
The in vivo antitumor effect of Gd-FLPNPs was evalu- treatment. There was no big difference in body weight loss
ated using nude mice bearing KB tumor xenografts, and after treatments for up to 14 days in the test groups, demon-
the tumor volumes of the mice were monitored every other strating that all treatments including iv injection of the agent
day. In the test group, mice were caudal vein injected with and drugs had good biocompatibility and biosafety.
submit your manuscript | www.dovepress.com
International Journal of Nanomedicine 2017:12
6879
Dovepress

Wu et al
Dovepress
$
%
*Gꢉ)/313V
*Gꢉ/313V
0DJQHYLVW
ꢇꢀꢀ
ꢆꢀ
ꢅꢀ
ꢄꢀ
ꢃꢀ
ꢀ
*Gꢉ)/313VꢊꢋLQꢊWKH
SUHVHQFHꢊRIꢊIRODWHꢌ
*Gꢉ/313V
*Gꢉ)/313V
)UHHꢊ'2;
ꢇꢀꢀ
ꢆꢀ
ꢅꢀ
ꢄꢀ
ꢃꢀ
ꢀ
ꢇꢀ
ꢃꢀ
ꢄꢀ
ꢅꢀ
ꢇꢀꢀ
ꢀ
ꢇ
ꢃ
ꢈ
ꢄ
ꢂ
&RQFHQWUDWLRQꢀꢁPJꢂ/ꢃ
&RQFHQWUDWLRQꢀRIꢀ'2;ꢀꢁ—JꢂP/ꢃ
ꢀꢁꢂ
ꢀꢁꢄ
ꢀꢁꢈ
ꢀꢁꢃ
ꢀꢁꢇ
ꢀꢁꢀ
&
'
ꢂꢀꢀ
ꢄꢀꢀ
ꢈꢀꢀ
ꢃꢀꢀ
ꢇꢀꢀ
3%6
)UHHꢊ'2;
*Gꢉ/313V
*Gꢉ)/313V
ꢀ
ꢃ
ꢄ
ꢅ
ꢆ
ꢇꢀ
ꢇꢃ
ꢇꢄ
*Gꢉ)/313V )UHHꢊ'2; *Gꢉ/313V
3%6
7LPHꢀꢁGD\Vꢃ
Figure 7 In vitro and in vivo cancer therapy in KB tumor-bearing mice.
Notes: (A) cell viability of KB cells incubated with non-DOX loaded gd-FlPNPs for 48 hours by MTT assay. (B) cytotoxicity of gd-FlPNPs, gd-lPNPs, gd-FlPNPs
pretreated with excess of folate (0.2 mM free folate acid was added to the medium), and free DOX against KB cells after incubation for 8 hours (n=4). (C) changes in the
relative tumor volume (n=4) after intravenous injection at days 0, 5, and 10 (DOX dosage: 6 mg/kg). *(P,0.1) and **(P,0.01). (D) relative tumor weight of the mice with the
various treatments.
Abbreviations: gd-FlPNPs, gd-coated folate-targeted lipid-polymer hybrid nanoparticles; DOX, doxorubicin; gd-lPNPs, gd-coated lipid-polymer hybrid nanoparticles;
MTT, 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-h-tetrazolium bromide; PBs, phosphate-buffered saline.
than non-targeted Gd-LPNPs, which can reduce side effects
Conclusion
of anticancer drugs at a desired location. In conclusion, the
multifunctional drug delivery system is a promising platform
for image-guided drug delivery systems.
In summary, we developed a multifunctional NP system
where the targeting ligand, CA, and anticancer drugs could
all be incorporated into one platform. We demonstrated
that Gd-FLPNPs have high monodispersity, excellent
size stability, and a well-defined core-shell structure.
In vitro release experiments showed that Gd-FLPNPs as a
reduction-sensitive drug deliver released DOX faster with
the presence of DTT than without it. In vitro and in vivo
MRI images showed that this type of NP had paramagnetic
properties with about two-fold enhancement in the longitu-
dinal relaxivity compared to the clinically used Magnevist.
That confirmed the potential of Gd-FLPNPs as an effective
T1-WI MRI CA. Furthermore, FCM, confocal image, and
MTT assay revealed that Gd-FLPNPs further enhanced cell
Acknowledgments
We thank Ping Yu for technical assistance, Lei Liu for the
illustrations and digital pictures, and Liu-Jie Zhang for cell
experiments. The authors acknowledge funding from the
National Natural Science Foundation of China (81372369,
51473127, 81571734 and 51673152), China Postdoctoral
Science Foundation (2017M612514), and Zhongnan Hospital
of Wuhan University Science, Technology and Innovation
Seed Fund Project (cxpy20160011).
Disclosure
uptake and produced higher cytotoxicity against KB cells The authors report no conflicts of interest in this work.
submit your manuscript | www.dovepress.com
International Journal of Nanomedicine 2017:12
6880
Dovepress

Dovepress
MrI-guided targeting delivery of doxorubicin
17. Singh R, Karakoti AS, Self W, Seal S, Singh S. Redox-sensitive
cerium oxide nanoparticles protect human keratinocytes from oxida-
tive stress induced by glutathione depletion. Langmuir. 2016;32(46):
12202–12211.
References
1. Weissleder R, Pittet MJ. Imaging in the era of molecular oncology.
Nature. 2008;452(7187):580–589.
2. Mi P, Cabral H, Kokuryo D, et al. Gd-DTPA-loaded polymer-metal
complex micelles with high relaxivity for MR cancer imaging.
Biomaterials. 2013;34(2):492–500.
3. Hubbell JA, Chilkoti A. Nanomaterials for drug delivery. Science. 2012;
337(6092):303–305.
4. Xu W, Burke JF, Pilla S, Chen H, Jaskula-Sztul R, Gong S. Octreotide-
functionalized and resveratrol-loaded unimolecular micelles for targeted
neuroendocrine cancer therapy. Nanoscale. 2013;5(20):9924–9933.
5. Floyd WC, Klemm PJ, Smiles DE, et al. Conjugation effects of various
linkers on Gd(III) MRI contrast agents with dendrimers: optimizing
the hydroxypyridinonate (HOPO) ligands with nontoxic, degradable
esteramide (EA) dendrimers for high relaxivity. J Am Chem Soc. 2011;
133(8):2390–2393.
6. Li X, Zhou H, Yang L, et al. Enhancement of cell recognition in vitro
by dual-ligand cancer targeting gold nanoparticles. Biomaterials. 2011;
32(10):2540–2545.
7. Cheng ZL, Tsourkas A. Monitoring phospholipase A2 activity with
Gd-encapsulated phospholipid liposomes. Sci Rep. 2014;4:6958.
8. Li XY, Wu MY, Pan LM, Shi JL. Tumor vascular-targeted co-delivery
of anti-angiogenesis and chemotherapeutic agents by mesoporous
silica nanoparticle-based drug delivery system for synergetic therapy
of tumor. Int J Nanomedicine. 2016;11:93–105.
18. Mura S, Nicolas J, Couvreur P. Stimuli-responsive nanocarriers for
drug delivery. Nat Mater. 2013;12(11):991–1003.
19. Wu B, Lu ST, Zhang LJ, Zhuo RX, Xu HB, Huang SW. Codelivery of
doxorubicin and triptolide with reduction-sensitive lipid-polymer hybrid
nanoparticles for in vitro and in vivo synergistic cancer treatment. Int J
Nanomedicine. 2017;12:1853–1862.
20. Vergote IB, Marth C, Coleman RL. Role of the folate receptor in ovar-
ian cancer treatment: evidence, mechanism, and clinical implications.
Cancer Metastasis Rev. 2015;34(1):41–52.
21. Satsangi A, Roy SS, Satsangi RK, et al. Synthesis of a novel, sequen-
tially active-targeted drug delivery nanoplatform for breast cancer
therapy. Biomaterials. 2015;59:88–101.
22. Shi D. Integrated multifunctional nanosystems for medical diagnosis
and treatment. Adv Funct Mater. 2009;19(21):3356–3373.
23. Pan J, Feng SS. Targeting and imaging cancer cells by folate-decorated,
quantum dots (QDs)-loaded nanoparticles of biodegradable polymers.
Biomaterials. 2009;30(6):1176–1183.
24. Kimpe K, Parac-Vogt Tatjana N, Laurent S, et al. Potential MRI
contrast agents based on micellar incorporation of amphiphilic
bis(alkylamide) derivatives of [(Gd−DTPA)(H2O)]2−. Eur J Inorg Chem.
2003;16:3021–3027.
25. Taratula O, Kuzmov A, Shah M, Garbuzenko OB, Minko T. Nano-
structured lipid carriers as multifunctional nanomedicine platform
for pulmonary co-delivery of anticancer drugs and siRNA. J Control
Release. 2013;171(3):349–357.
9. Li L, ten Hagen TLM, Haeri A, et al. A novel two-step mild hyper-
thermia for advanced liposomal chemotherapy. J Control Release. 2014;
174:202–208.
10. Wang S, Wang H, Liu Z, et al. Smart pH- and reduction-dual-responsive
folate-PEG-coated polymeric lipid vesicles for tumor-triggered targeted
drug delivery. Nanoscale. 2014;6(13):7635–7642.
26. Maeda H, Wu J, Sawa T, Matsumura Y, Hori K. Tumor vascular perme-
ability and the EPR effect in macromolecular therapeutics: a review.
J Control Release. 2000;65(1):271–284.
11. Karimi M, Ghasemi A, Sahandi Zangabad P, et al. Smart micro/
nanoparticles in stimulus-responsive drug/gene delivery systems. Chem
Soc Rev. 2016;45(5):1457–1501.
12. El-Dakdouki MH, El-Boubbou K, Kamat M, et al. CD44 targeting
magnetic glyconanoparticles for atherosclerotic plaque imaging.
Pharm Res. 2014;31(6):1426–1437.
13. Wu B, Yu P, Cui C, et al. Folate-containing reduction-sensitive lipid-
polymer hybrid nanoparticles for targeted delivery of doxorubicin.
Biomater Sci. 2015;3(4):655–664.
14. Cui C, Xue YN, Wu M, et al. Cellular uptake, intracellular traffick-
ing, and antitumor efficacy of doxorubicin-loaded reduction-sensitive
micelles. Biomaterials. 2013;34(15):3858–3869.
15. Zhang LJ, Wu B, Zhou W, et al. Two-component reduction-sensitive
lipid-polymer hybrid nanoparticles for triggered drug release and
enhancedinvitroandinvivoanti-tumorefficacy. BiomaterSci. 2016;5(1):
98–110.
16. Chen W, Zhong P, Meng F, et al. Redox and pH-responsive degrad-
able micelles for dually activated intracellular anticancer drug release.
J Control Release. 2013;169(3):171–179.
27. Fang J, Nakamura H, Maeda H. The EPR effect: unique features of
tumor blood vessels for drug delivery, factors involved, and limita-
tions and augmentation of the effect. Adv Drug Deliv Rev. 2011;63(3):
136–151.
28. Oh JM, Choi SJ, Kim ST, Choy JH. Cellular uptake mechanism of
an inorganic nanovehicle and its drug conjugates: enhanced efficacy
due to clathrin-mediated endocytosis. Bioconjug Chem. 2006;17(6):
1411–1417.
29. SolomonI.Relaxationprocessesinasystemoftwospins.Phys Rev. 1955;
99(2):559–565.
30. Jung SH, Na K, Lee SA, Cho SH, Seong H, Shin BC. Gd(III)-DOTA-
modified sonosensitive liposomes for ultrasound-triggered release and
MR imaging. Nanoscale Res Lett. 2012;7(1):462.
31. Hak S, Sanders HMHF, Agrawal P, et al. A high relaxivity Gd(III)
DOTA-DSPE-based liposomal contrast agent for magnetic resonance
imaging. Eur J Pharm Biopharm. 2009;72(2):397–404.
32. Kielar F, Tei L, Terreno E, Botta M. Large relaxivity enhancement of
paramagnetic lipid nanoparticles by restricting the local motions of the
GdIII chelates. J Am Chem Soc. 2010;132(23):7836–7837.
submit your manuscript | www.dovepress.com
International Journal of Nanomedicine 2017:12
6881
Dovepress

Wu et al
Dovepress
Supplementary materials
G
R
J
2
F
I
G
E
L
V
R
R
5
2+
R
R
V
M
ꢇ
2+
+2
ꢅ
L
J
K
I
H
G
P
D
R
5
5
2
'&&ꢀ'0$3
2
5
2
+2
L
2
&
'&&ꢀ'0$3
2
ꢂꢃꢁ5ꢁ5ꢁ&22+
2
2+
P
2
P3(*
H
2
5
I
M
J
E
D
P3(*
F
K
5
2
2
P3(*ꢁ5ꢁ5ꢁ&ꢂꢃ
ꢀꢁꢂ ꢀꢁꢃ ꢀꢁꢄ ꢃꢁꢅ ꢃꢁꢆ ꢇꢁꢂ ꢇꢁꢃ ꢇꢁꢄ ꢆꢁꢅ ꢆꢁꢆ ꢈꢁꢂ ꢈꢁꢃ ꢈꢁꢄ ꢄꢁꢅ SSP
ꢁ5ꢁ5ꢁꢄ
ꢁ6ꢁ6ꢁꢅꢄP3(*ꢁ6ꢁ6ꢁ&ꢂꢃ
ꢁ&+ꢆ&+ꢆꢅꢄP3(*ꢁ&ꢁ&ꢁ&ꢂꢃ
Figure S2 1h NMr spectra of mPeg-s-s-c16 polymer in cDcl3 (ppm).
Abbreviations:mPeg-s-s-c16,monomethoxy-poly-(ethyleneglycol)-s-s-hexadecyl;
NMr, nuclear magnetic resonance.
Figure S1 synthesis pathway of the amphiphilic polymers (mPeg-s-s-c16).
Abbreviations: Dcc, dicyclohexylcarbodiimide; DMaP, 4-dimethylaminopyridine;
mPeg-s-s-c16, monomethoxy-poly-(ethylene glycol)-s-s-hexadecyl.
International Journal of Nanomedicine
Dovepress
Publish your work in this journal
The International Journal of Nanomedicine is an international, peer-
reviewed journal focusing on the application of nanotechnology
in diagnostics, therapeutics, and drug delivery systems throughout
the biomedical field. This journal is indexed on PubMed Central,
MedLine, CAS, SciSearch®, Current Contents®/Clinical Medicine,
Journal Citation Reports/Science Edition, EMBase, Scopus and the
Elsevier Bibliographic databases. The manuscript management system
is completely online and includes a very quick and fair peer-review
system, which is all easy to use. Visit http://www.dovepress.com/
testimonials.php to read real quotes from published authors.
Submit your manuscript here: http://www.dovepress.com/international-journal-of-nanomedicine-journal
submit your manuscript | www.dovepress.com
International Journal of Nanomedicine 2017:12
6882
Dovepress