PEG-detachable lipid-polymer hybrid nanoparticle for delivery of chemotherapy drugs to cancer cells

Article abstract of DOI:10.1097/CAD.0000000000000092

The experiment aimed to increase the drug-delivery efficiency of poly-lactic-co-glycolic acid (PLGA) nanoparticles. Lipid-polymer hybrid nanoparticles (LPNs-1) were prepared using PLGA as a hydrophobic core and FA-PEG-hyd-DSPE as an amphiphilic shell. Uniform and spherical nanoparticles with an average size of 185 nm were obtained using the emulsification solvent evaporation method. The results indicated that LPNs-1 showed higher drug loading compared with naked PLGA nanoparticles (NNPs). Drug release from LPNs-1 was faster in an acidic environment than in a neutral environment. LPNs-1 showed higher cytotoxicity on KB cells, A549 cells, MDA-MB-231 cells, and MDA-MB-231/ADR cells compared with free doxorubicin (DOX) and NNPs. The results also showed that, compared with free DOX and NNPs, LPNs-1 delivered more DOX to the nuclear of KB cells and MDA-MB-231/ADR cells. LPNs-1 induced apoptosis in KB cells and MDA-MB-231/ADR cells in a dose-dependent manner. The above data indicated that DOX-loaded LPNs-1 could kill not only normal tumor cells but also drug-resistant tumor cells. These results indicated that modification of PLGA nanoparticles with FA-PEG-hyd-DSPE could considerably increase the drug-delivery efficiency and LPNs-1 had potential in the delivery of chemotherapeutic agents in the treatment of cancer.

Full text of DOI:10.1097/CAD.0000000000000092

Preclinical report 751
PEG-detachable lipid–polymer hybrid nanoparticle for
delivery of chemotherapy drugs to cancer cells
Jiang-bo Du*, Yan-feng Song*, Wei-liang Ye, Ying Cheng, Han Cui,
Dao-zhou Liu, Miao Liu, Bang-le Zhang and Si-yuan Zhou
The experiment aimed to increase the drug-delivery
efficiency of poly-lactic-co-glycolic acid (PLGA)
tumor cells. These results indicated that modification
of PLGA nanoparticles with FA-PEG-hyd-DSPE could
considerably increase the drug-delivery efficiency and
LPNs-1 had potential in the delivery of chemotherapeutic
agents in the treatment of cancer. Anti-Cancer Drugs
nanoparticles. Lipid–polymer hybrid nanoparticles (LPNs-1)
were prepared using PLGA as a hydrophobic core and
FA-PEG-hyd-DSPE as an amphiphilic shell. Uniform
and spherical nanoparticles with an average size of 185 nm
were obtained using the emulsification solvent evaporation
method. The results indicated that LPNs-1 showed higher
drug loading compared with naked PLGA nanoparticles
(NNPs). Drug release from LPNs-1 was faster in an acidic
environment than in a neutral environment. LPNs-1 showed
higher cytotoxicity on KB cells, A549 cells, MDA-MB-231
cells, and MDA-MB-231/ADR cells compared with free
doxorubicin (DOX) and NNPs. The results also showed
that, compared with free DOX and NNPs, LPNs-1 delivered
more DOX to the nuclear of KB cells and MDA-MB-231/
ADR cells. LPNs-1 induced apoptosis in KB cells and
MDA-MB-231/ADR cells in a dose-dependent manner.
The above data indicated that DOX-loaded LPNs-1 could
kill not only normal tumor cells but also drug-resistant
ꢀc
25:751–766
2014 Wolters Kluwer Health | Lippincott
Williams & Wilkins.
Anti-Cancer Drugs 2014, 25:751–766
Keywords: doxorubicin, folic acid, lipid–polymer hybrid nanoparticles,
multidrug resistance
Department of Pharmaceutics, School of Pharmacy, Fourth Military Medical
University, Xi’an, China
Correspondence to Si-yuan Zhou, PhD, Department of Pharmaceutics, School
of Pharmacy, Fourth Military Medical University, Changle West Road 17,
Shaanxi province, Xi’an 710032, China
Tel: + 86 29 84776783; fax: + 86 29 84779212; e-mail: zhousy@fmmu.edu.cn
*Jiang-bo Du and Yan-feng Song contributed equally to the writing of this article.
Received 2 October 2013 Revised form accepted 24 January 2014
Introduction
conjugated with polyethylene glycol (PEG) have been
grafted onto the polymeric nanoparticles to form lipid–
polymer hybrid nanoparticles (LPNs) [12–17]. PEG
causes a steric hindrance against the opsonin adsorption
because of its hydrophilicity and brush structure on the
surface of nanoparticle. As a result, PEGylated polymeric
nanoparticles have shown the following characteristics:
(a) prolonged in-vivo blood circulation time, (b) in-
creased aqueous solubility and stability, (c) reduced
aggregation, and (d) attenuated immunogenicity [18–22].
Polymeric nanoparticles are used widely in drug-delivery
systems as they have high structural integrity, stability
during storage, and controlled-release property. Besides,
they can be prepared easily and functionalized into an
active targeted drug-delivery system. These characteristics
make them highly attractive as chemotherapeutic drug-
delivery carriers [1,2]. Polymeric nanoparticles can be
prepared from both natural polymers (such as chitosan) and
synthetic biodegradable and biocompatible polymers (such
as poly-lactic-co-glycolic acid, PLGA). PLGA nanoparticles
were used as an effective nanocarrier for the encapsulation
of various anticancer agents such as paclitaxel [3],
9-nitrocamptothecin [4], and cisplatin [5], and also for
the encapsulation of haloperidol and estradiol [6].
As an effective drug-delivery system, nanoparticles should
disassemble and release drug in an efficient manner after it
is localized in pathological tissues. The PEG coating,
however, reduces the interactions between lipid-based
nanoparticles and biological membrane, which result in
delay of drug release and decrease in therapeutic efficacy
[23,24]. Moreover, contact of PEGylated nanoparticles with
immune cells for a long duration of time can induce the
generation of a PEG-specific antibody, which significantly
decreases the half-life of the subsequent doses of PEGylated
nanoparticles [25,26]. This can help to explain why some
PEGylated nanoparticles have shown accelerated blood
clearance phenomena (ABC phenomena). Therefore, de-
PEGylation is needed to enhance the drug release at the
target site and the clearance of the PEGylated nanoparticles
However, systematic administration of the bare polymeric
nanoparticles cannot be performed because they are not
stable in blood circulation and can be uptaken rapidly by
the reticuloendothelial system [7–10]. Macrophage cells
such as the Kupffer cells in the liver play a major role in
the clearance mechanism. These macrophages cannot
identify the nanoparticles directly; however, they can
recognize the specific opsonin adsorbed on the surface
of the nanoparticles [11]. To reduce the uptake of
nanoparticles by reticuloendothelial system, various lipids
ꢀc
0959-4973
2014 Wolters Kluwer Health | Lippincott Williams & Wilkins
DOI: 10.1097/CAD.0000000000000092
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752 Anti-Cancer Drugs 2014, Vol 25 No 7
from the circulation. A balance between PEGylation and de-
PEGylation should be maintained to produce a useful and
safe nanoparticle formulation [27–30].
Methods
Preparation of FA-PEG-hyd-DSPE
The synthesis scheme for FA-PEG-hyd-DSPE is shown
in Fig. 1.
In principle, the PEG shedding approaches are categor-
ized as diffusible PEG conjugates and connection with a
degradable linker [30]. Among these, the promising
approach to shed PEG is using a linker with a
predetermined cleavage point between the PEG chain
and the lipid moiety. Because the nanoparticles enter cells
through the endocytosis pathway and usually localize in
endolysosomes where pH decreases to 5.5–6.0 in endo-
somes and 4.5–5.0 in lysosomes, and a redox potential
exists between the extracellular space and the endosomal
environment because of the rich glutathione inside the
endosome [31,32] thus, chemical stimuli, such as low pH
or reducing agents, have been exploited to break the
linker [33–36]. Enzymatic stimuli, such as proteases, have
also been explored to induce the cleavage of PEG [37].
Synthesis of FA-PEG-COOH: FA (221 mg), DCC
(206 mg), and NHS (115 mg) were dissolved in 4 ml
dimethylsulfoxide (DMSO) and stirred at room tempera-
ture for 5 h. The stirred solution was added dropwise into
an NH2-PEG-COOH containing (100 mg) DMSO solu-
tion and stirred at room temperature overnight. Then,
the mixture was filtered to remove N,N0-dicyclohexylurea.
The filtrate was diluted with 10-fold volume of water and
dialyzed (molecular weight cut-off: 1000) in water for 2
days before it was collected by freeze-drying. The
product was dissolved in water and the solution was
further purified by Sephadex G-25. The target compo-
nent was lyophilized to produce a yellow solid powder
[38–40].
In this experiment, we have synthesized a pH-sensitive
amphiphilic block copolymer: folic acid-poly(ethyleneglycol)-
2-distearoyl-sn-glycero-3-phosphoethanolamine (FA-PEG-
hyd-DSPE), in which DSPE was connected to PEG
by a hydrazone bond and FA was a targeting moiety.
FA-PEG-hyd-DSPE was used to decorate PLGA nano-
particles to form LPNs with a core–shell structure. It is
anticipated that these pH-sensitive LPNs can increase
drug loading, shed PEG in acidic organelles in tumor
cells, and release the chemotherapy drug with high
efficiency.
Synthesis of FA-PEG-NH-NH-BOC: FA-PEG-COOH
(100 mg), DCC (8.2 mg), and DMAP (0.2 mg) were
dissolved in 2 ml DMSO and reacted for 5 h. Then,
NH2-NH-BOC (6 mg) was added to the reaction solu-
tion. After stirring at room temperature overnight, the
mixture was filtered to remove N,N-dicyclohexylurea.
The filtrate was dialyzed in water for 2 days and then
collected by freeze-drying.
Synthesis of FA-PEG-NH-NH2 FA-NH-NH-BOC (50 mg)
was dissolved in dichloromethane and 2 ml trifluoroacetic
acid was added. The mixture was reacted at room
temperature for 2 h. The solvent was removed using a
vacuum rotary evaporator. The residue was dialyzed and
purified by Sephadex G-25 as described above.
Materials and methods
Materials
PLGA (lactic/glycolic acid molar ratio 75/25 and average
molecular weight 40–75 kDa) was obtained from EVONIK
Industries (Essen, Germany). Polyvinyl alcohol (molecular
weight: 20 000–30 000) was purchased from Shanghai
Sangon Biological Engineering Technology & Services Co.
Ltd (Shanghai, China). 1,2-Distearoyl-sn-glycero-3-phos-
phoethanolamine (DSPE) was purchased from J&K Chemi-
ca (Beijing, China). Folic acid (FA), N-hydroxysuccinimide
(NHS), dicyclohexylcarbodiimide (DCC), trifluoroacetic
acid, and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazo-
lium bromide (MTT) were purchased from Sigma-Aldrich
Company (St Louis, Missouri, USA). a-Carboxyl-o-amino
poly (ethylene glycol) (HOOC-PEG-NH2, average molecu-
lar weight 4000) was obtained from Shanghai Yare Biotech
Inc. (Shanghai, China). Doxorubicin (DOX) was purchased
from Hisun Pharmaceutical Co. (Zhejiang, China). The
human squamous carcinoma cell (KB cell, overexpressing
folate receptor), human breast cancer cell (MDA-MB-231
cell), and the folate receptor deficient human lung
adenocarcinoma epithelial cell (A549 cell) were obtained
from the Institute of Biochemistry and Cell Biology, Chinese
Academy of Science (Shanghai, China). The DOX-resistant
cell, MDA-MB-231/ADR, was prepared in our laboratory.
Synthesis of DSPE-4-acetylbenzoic acid: 4-acetylbenzoic
acid (30 mg), DCC (28.8 mg), and NHS (16.1 mg) were
dissolved in 2 ml dichloromethane. The mixture was
reacted at room temperature for 6 h before DSPE (70 mg)
and triethylamine were added to the solution. After
stirring at room temperature for another 6 h, the product
was extracted by adding water and dichloromethane in
the reaction mixture. The organic phase was dried by
Na2SO4 and evaporated using a vacuum rotary evaporator.
The residue was purified by silica gel chromatography.
Synthesis of FA-PEG-hyd-DSPE FA-PEG-NH-NH2
(50 mg) and DSPE-acetyl benzoic acid (20 mg) were
dissolved in 2 ml DMSO and reacted in the presence of
trifluoroacetic acid at room temperature for 24 h. The
reaction mixture was diluted with 10-fold volume of
water. The solution was dialyzed and further purified by
Sephadex G-25 as described above.
NH2-PEG-hyd-DSPE NH2-PEG-hyd-DSPE was synthe-
sized using the same methods as FA-PEG-hyd-DSPE
using NH2-PEG-NH-NH2 as a substrate. FA-PEG-hyd-
DSPE was used as a control.
Copyright © Lippincott Williams & Wilkins. Unauthorized reproduction of this article is prohibited.

PEG-detachable lipid–polymer hybrid nanoparticle Du et al. 753
Fig. 1
OH
OH
N
O
HN
N
O
N:C:N
N
N
N
N
H
COOH
N
N
H
COOH
COOH
HN
H2N
N
N
H2N
N
O
C
NH
C
Folate
Activated folate
O
OH
N
O
N
N
N
N
H
COOH
H
NH2-PEG-COOH
HN
H2N
O
C
O
N
O
C
OH
FA-PEG-COOH
n
OH
N
O
O
NH2
N
N
N
N
H
COOH
O
N
H
HN
H2N
H
N
O
O
C
H
O
N
C
O
N
n
O
H
FA-PEG-NH-NH-BOC
COOH
HO
O
N
N
N
N
TFA
H
N
H
H2N
N
O
H
H
O
N
C
C
N
NH2
n
O
FA-PEG-NH-NH2
O
O
P
O
O
O
O
HOOC
C CH3
OH
NH2
H
O
O
O
O
FA-PEG-NH-NH2
P
O
O
O
O
O
OH
N
H
C
C CH3
H
O
O
O
O
P
O
O
O
O
C
OH
N
H
O
H
O
OH
O
N
N
COOH
H
C
N
CH3
N
N
H
HN
H2N
N
HN
C
O
N
O
n
FA-PEG-hyd-DSPE
Synthetic scheme of the FA-PEG-hyd-DSPE conjugate. FA-PEG-hyd-DSPE, folic acid-poly(ethyleneglycol)-2-distearoyl-sn-glycero-3-
phosphoethanolamine.
Preparation of LPNs
reported method [41]. In brief, 16.0 mg PLGA and
6.0 mg FA-PEG-hyd-DSPE were dissolved in 8 ml
methylene dichloride, 15 ml of 3.0% polyvinyl alcohol
aqueous solution containing 3.0 mg DOX was added,
The lipid–polymer hybrid PLGA nanoparticles (LPNs-1)
and naked PLGA nanoparticles without FA-PEG-hyd-
DSPE (NNPs) were prepared using the previously
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754 Anti-Cancer Drugs 2014, Vol 25 No 7
and the resulting mixture solution was sonicated in a
25 ml round-bottom flask for 30 s using a probe sonication
power of 400 W in an ice bath. After stirring at room
temperature for 4 h, the LPNs-1 were collected by
centrifugation at 8000g (10 min) and the precipitate was
washed three times by deionized water. LPNs-2 was
prepared using the same methods as LPNs-1 using NH2-
PEG-hyd-DSPE as an amphiphilic shell.
Cytotoxicity assay
To evaluate the cytotoxicity of the free DOX and DOX-
loaded LPNs or DOX-loaded NNPs, KB cells, MDA-MB-
231 cells, MDA-MB-231/ADR cells, and A549 cells were
used as in-vitro models. Cells were seeded in 96-well plates
(10 000 cells/per well) and incubated for 12 h. Then, the
cells were exposed to different concentrations of free DOX
or DOX-loaded nanoparticles. Forty-eight hours after drug
treatment, 20 ml of the MTTsolution (5 mg/ml) was added
and incubated for 4 h at 371C. The medium was replaced
with 150 ml of DMSO. The absorbance was measured at
490 nm using a Bio-Rad Microplate Reader (Bio-Rad
Laboratories, Richmond, California, USA). The cytotoxicity
of DOX or DOX-loaded nanoparticles was shown as a cell
viability percentage against the respective control [43].
Size and morphology of LPNs and NNPs
The particle size, polydispersity index, and B potential of
LPNs and NNPs were determined at 251C by dynamic
light scattering using a Beckman Coulter Particle
Analyzer (Fullerton, California, USA). Three different
samples were prepared, measured, and the data were
averaged. The morphology of nanoparticles was observed
using transmission electron microscopy (JEOL-100CXII;
JEOL, Tokyo, Japan). A drop of the sample was deposited
onto a carbon-coated copper grid to create a thin film.
Before the film was dried, it was counterstained with 2%
phosphotungstic acid by adding a drop of the staining
solution to the film. The excess solution was drained by
filter paper. The grid was allowed to dry at room
temperature and the sample was then determined under
transmission electron microscopy.
Evaluation of cellular uptake of LPNs-1 and NNPs
DOX shows red fluorescence, which can be used directly to
investigate its cellular uptake. Cells were seeded into
coverglass-containing 24-well plates at a density of 100 000
cells/well and incubated at 371C for 12h. DOX or DOX-
loaded LPNs-1 (10 mg/ml equivalent DOX) was added and
incubated for 4 h at 371C. The cells were then washed five
times with PBS and treated with 40,6-diamidino-2-phenyl-
indole (10 mg/ml) for 15 min for nucleus staining. Then, the
cells were washed with PBS three times and fixed with 1.5%
formaldehyde. Cover slips were placed onto glass micro-
scope slides and DOX uptake was analyzed using confocal
laser scanning microscopy (Leica, Wetzler, Germany).
Surface chemistry of LPNs and NNPs
The surface compositions of the DOX-loaded NNPs,
DOX-free LPNs-1, and DOX-loaded LPNs-1 were
investigated using an X-ray photoelectric spectrometer
(XPS; RATOS AXIS His system, Shimadzu, Japan). The
binding energy spectrum was analyzed from 0 to 1100 eV
in a fixed transmission mode with a passing energy of
80 eV [42].
Cellular uptake of DOX or DOX-loaded LPNs-1 in KB cells
and MDA-MB-231/ADR cells was also determined semi-
quantitatively using flow cytometry (Coulter XL, Beckman;
Beckman-Coulter, Hialeah, Florida, USA). Briefly, KB cells
(or MDA-MB-231/ADR cells) were seeded into 24-well
plates at a cell density of 1 ꢁ 105 cells/ml. After 24 h, the
medium was removed and fresh medium containing 10 mg/ml
free DOX or DOX-loaded LPNs-1 was added (200 ml each
well) and incubated with the cells at 371C for 30min or 3h.
The cells were collected in PBS and centrifuged for 2 min at
1000g to remove the supernatant. Finally, the cells were
resuspended in 0.2ml PBS and analyzed by flow cytometry.
In-vitro drug-release study
Ten milligram of freshly prepared DOX-loaded LPNs or
DOX-loaded NNPs was dispersed in 10 ml PBS and
incubated in a water bath shaker at 371C. At appropriate
intervals, 0.2 ml supernatant was withdrawn after cen-
trifugation at 8000g and the same volume of fresh
medium was supplemented. The DOX released was
quantified by fluorescence spectroscopy (970 CRT
Spectrofluorophotometer; Shanghai Precision and Scientific
Instrument Co. Ltd, Shanghai, China).
Apoptosis analysis
MDA-MB-231/ADR cells and KB cells were seeded into
six-well plates at a cell density of 5 ꢁ 106 cells/ml in RPMI
1640 medium. After 24 h, the medium was removed and
fresh medium containing DOX or DOX-loaded LPNs-1 was
added and incubated with the cells at 371C for 24 h.
Subsequently, the medium was removed and cells were
washed three times with PBS, and then the cells were
suspended in PBS and centrifuged for 2 min at 1000g to
remove the supernatant, and the cells were resuspended in
0.2 ml PBS. After staining with annexin V-FITC and
propidium iodide, the cells were analyzed by Becton
Dickinson FACScan (excitation at 488 nm) (Becton
Dickinson Corp., San Jose, California, USA).
Cell culture conditions
KB cells were maintained in a folate-free RPMI 1640
medium. MDA-MB-231 cells, MDA-MB-231/ADR cells,
and A549 cells were maintained in an RPMI 1640
medium. The cells were supplemented with 100 U/ml
penicillin, 100 U/ml streptomycin, and 10% fetal bovine
serum. The cells were cultured as a monolayer in a
humidified atmosphere containing 5% CO2 at 371C.
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PEG-detachable lipid–polymer hybrid nanoparticle Du et al. 755
Fig. 2
110
107.5
105
102.5
100
97.5
95
92.5
90
87.5
85
82.5
80
77.5
75
72.5
70
67.5
65
62.5
60
57.5
55
52.5
50
3400
3200
3000
2800
2600
2400
2200
2000
1/cm
1800
1600
1400
1200
1000 900 800 700
500
400
300
200
100
15.0
10.0
5.0
0.0
ppm (f1)
FTIR spectrum and 1H NMR spectrum (dissolved in dimethylsulfoxide) of the FA-PEG-hyd-DSPE conjugate. FA-PEG-hyd-DSPE, folic acid-
poly(ethyleneglycol)-2-distearoyl-sn-glycero-3-phosphoethanolamine.
Results and discussion
showed characteristic absorbance for PEG as the C-O-C
etheric bond bending vibration at 1113/cm and absorption
at 774/cm attributable to C-H bond vibration on the
benzene ring. The absorption at 1662/cm was attributable
Characterization of the FA-PEG-hyd-DSPE conjugate
1
The FTIR spectrum and H NMR spectrum of FA-PEG-
hyd-DSPE are presented in Fig. 2. FA-PEG-hyd-DSPE
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756 Anti-Cancer Drugs 2014, Vol 25 No 7
Fig. 3
120
115
110
105
100
95
90
85
80
75
70
65
60
1/cm
900
800
700
600
500
400
300
200
100
ppm (f1)
FTIR spectrum and 1H NMR spectrum (dissolved in CHCl3) of the PEG-hyd-DSPE conjugate.
Copyright © Lippincott Williams & Wilkins. Unauthorized reproduction of this article is prohibited.

PEG-detachable lipid–polymer hybrid nanoparticle Du et al. 757
Fig. 4
Intensity distribution
NNPs
NNPs
10
250
200
150
100
50
0.5
0.4
0.3
0.2
0.1
0.0
100
75
Diameter
PDI
NNPs
50
25
0
5
0
0
1.0
10.0
100.0
1000.0
1
2
3
4
5
6
7
8
Diameter (nm)
Time (days)
Intensity distribution
LPNs-1
LPNs-1
10
250
200
150
100
50
0.5
0.4
0.3
0.2
0.1
0.0
100
75
50
25
0
LPNs-1
Diameter
PDI
5
0
0
1.0
10.0
100.0
1000.0
1
2
3
4
5
6
7
8
Diameter (nm)
Time (days)
Transmission electron microscopy image, size distribution, and stability of LPNs-1 and NNPs. NNPs, naked PLGA nanoparticles;
LPNs-1, FA-PEG-hyd-DSPE modified PLGA nanoparticles.
Table 1 Characteristics of nanoparticles
Particle size (nm)
Polydispersity
B potential (mV)
Drug load (%)
Encapsulation efficiency (%)
LP1/PLGA = 0/16 (mg/mg)
LP1/PLGA = 1/16 (mg/mg)
LP1/PLGA = 3/16 (mg/mg)
LP1/PLGA = 5/16 (mg/mg)
LP1/PLGA = 7/16 (mg/mg)
LP2/PLGA = 5/16 (mg/mg)
165 13
173 18
182 17
185 12
213 20
202 10
0.10 0.02
0.13 0.03
0.13 0.04
0.12 0.03
0.15 0.04
0.14 0.02
– 34.5 2.7
– 29.5 1.6
– 26.3 1.2
– 25.7 1.1
– 25.8 2.7
– 16.9 1.7
2.2 0.3
3.3 0.4
7.8 0.7
9.2 2.3
9.3 3.0
6.2 1.3
26.7 7.3
48.9 8.9
64.3 6.2
76.9 8.1
77.7 8.5
56.9 6.7
LP1, FA-PEG-hyd-DSPE; LP2, PEG-hyd-DSPE; PLGA, poly-lactic-co-glycolic acid.
to stretching of the C = N bond of the hydrazone link.
The presence of FA in FA-PEG-hyd-DSPE was confirmed
by the appearance of signals at 7.1 and 7.8 ppm in the
1H NMR spectrum, which corresponded with the aro-
matic protons of FA. Moreover, the PEG backbone was
confirmed by the signal at 3.6 ppm. The FTIR spectrum
and 1H NMR spectrum of PEG-hyd-DSPE are shown
in Fig. 3.
hydrophobic DOX is more easily entrapped in the
hydrophobic core of the nanoparticles than the hydro-
philic salt form DOXꢂHCl [42].
The morphology and stability of LPNs-1 and NNPs are
shown in Fig. 4. The particle size, polydispersity index, B
potential, drug loading, and encapsulation efficiency of
LPNs and NNPs are shown in Table 1. The micrograph
of LPNs-1 and NNPs showed individual nanometric
particles. Both LPNs-1 and NNPs remained stable for
more than 8 days in PBS.
Particle characterization
When DOX-loaded nanoparticles were prepared, DOX
was deprotonated by adding an excess amount of
triethylamine to become hydrophobic. This is because
The B potential of nanoparticle is a significant parameter
as it plays an important role in the interaction between
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758 Anti-Cancer Drugs 2014, Vol 25 No 7
cells and nanoparticles as well as suspension stability.
In our experiment, the B potential of LPNs-1 ranged from
– 25.7 to – 29.5 mV, whereas the B potential of NNPs was
– 34.5 mV. The B potential of LPNs-1 shifted toward the
positive after the attachment of FA-PEG-hyd-DSPE on
the surface of NNPs, which resulted from the nitrogen
atoms in FA and DSPE moiety. It is established that
neutral or negatively charged nanoparticles could reduce
plasma protein adsorption and decrease the nonspecific
cellular uptake [44,45]. However, highly positive charged
nanoparticles could be removed easily from blood
circulation and were not favorable for tumor targeting.
Decreased nonspecific phagocytosis led to an increase in
circulation time, which allowed more nanoparticles to
accumulate in the tumor tissue to recognize the target
cells [16]. Furthermore, the greater the B potential of
nanoparticles, the more stable the nanoparticles suspen-
sion. This was because the charged particles repelled one
another and thus overcame the natural tendency to
aggregate.
(> 200 nm) tended to be cleared faster in the blood, and
more particles distributed in the liver, lung, and spleen.
Smaller nanoparticles (10–200 nm) with less negative
surface charge tended to accumulate in tumors while
being cleared from the blood at a slower rate [48,49].
Besides, the degree of leakiness of the tumor blood
vessel and the optimal size of the nanoparticles varied
significantly among different tumor types [47]. For
example, the vasculatures in human brain, pancreatic,
and ovarian cancers is less leaky than those of other
cancers [50,51].
Surface chemistry
The surface chemistry of LPNs-1 and NNPs is shown
in Fig. 5. The chemical composition weight percentages
of nitrogen on the surface of DOX-loaded NNPs,
DOX-free LPNs-1, and DOX-loaded LPNs-1 were
calculated to be 0, 0.49, and 2.31%, respectively. There
was one nitrogen atom in the DOX chemical structure
but no N 1s signal was observed for the DOX-loaded
NNPs (Fig. 5a), which indicated that DOX were well
encapsulated inside the NNPs. Nevertheless, there were
no nitrogen atoms in the chemical structure of PLGA and
there were 11 nitrogen atoms in the FA-PEG-hyd-DSPE
conjugate. There was an N 1s signal in the XPS data for
DOX-free LPNs-1 (Fig. 5b), and this indicated that folate
was coated on the surface of the LPNs-1 [42,52]. Thus,
the DOX-loaded LPNs-1 was guided to recognize the FA
high-expressed tumor cells. The N 1s percentage was
found to increase to 2.31% in the DOX-loaded LPNs-1
(Fig. 5c), which indicated that some amount of DOX was
encapsulated in the shell of the LPNs-1, composed of FA-
PEG-hyd-DSPE. This is the main reason why LPNs-1
showed enhanced drug-loading capacity and encapsula-
tion efficiency compared with NNPs. This discovery is
consistent with the previously reported studies [53–55].
The particle size, drug loading, and encapsulation effi-
ciency of LPNs-1 increased with an increase in the content
of FA-PEG-hyd-DSPE. The drug loading and encapsulation
efficiency of NNPs was, respectively, 2.2 and 26.7%. When
the ratio between FA-PEG-hyd-DSPE and PLGA was 6 : 16
(mg/mg), the particle size of LPNs-1 was 185 12 nm; the
drug loading and encapsulation efficiency could reach,
respectively, 9.2 and 76.9%. This indicated that modifica-
tion of PLGA nanoparticles with FA-PEG-hyd-DSPE not
only enabled active drug delivery and long circulation but
also enhanced the drug-delivery efficiency.
As is well known, particle size is a critical parameter that
affects the biodistribution of nanoparticles in the body.
The major pathway of extravasation for nanoparticles is
through the leakage of blood vessels. The enhanced
permeability and retention is an important feature of
tumor tissue, which allows nanoparticles (up to 400 nm)
to preferentially accumulate in tumor tissues [46,47]. It
was also reported that larger polymeric nanoparticles
In-vitro DOX release kinetics
In-vitro drug-release kinetics from DOX-loaded LPNs-1,
DOX-loaded LPNs-2, and DOX-loaded NNPs are shown
Fig. 5
(a)
(b)
(c)
Survey
Survey
Survey
2.00E+05
1.50E+05
1.00E+05
5.00E+04
0.00E+00
2.00E+05
1.50E+05
1.00E+05
5.00E+04
0.00E+00
1.50E+05
1.00E+05
5.00E+04
0.00E+00
C1s
C1s
C1s
O1s
O1s
O1s
N1s
N1s
1200 1000 800 600 400 200
Binding energy (eV)
0
1200 1000 800 600 400 200
Binding energy (eV)
0
1200 1000 800 600 400 200
Binding energy (eV)
0
The X-ray photoelectric spectrometer wide scan spectra of the DOX-loaded NNPs (a), DOX-free LPNs-1 (b), and DOX-loaded LPNs-1 (c).
DOX, doxorubicin; LPNs-1, FA-PEG-hyd-DSPE modified PLGA nanoparticles; NNPs, naked PLGA nanoparticles.
Copyright © Lippincott Williams & Wilkins. Unauthorized reproduction of this article is prohibited.

PEG-detachable lipid–polymer hybrid nanoparticle Du et al. 759
Fig. 6
(a)
Fig. 7
(a)
pH=7.4
pH=6.5
pH=5.0
120
50
45
40
35
30
25
20
15
10
100
80
60
40
20
0
0
10 20 30 40 50 60 70 80 90 100 110
0
0.05
0.1
0.5
1
(b)
85
80
75
70
65
60
55
50
45
40
35
30
25
20
15
10
(b)
120
100
80
60
40
20
0
0
10 20 30 40 50 60 70 80 90 100 110
(c)
85
80
75
70
65
60
55
50
45
40
35
30
25
20
15
10
0
0.05
0.1
0.5
1
Concentration (mg/ml)
The cytotoxicity of DOX-free LPNs-1 toward KB cells (a) and MDA-MB-
231 cells (b). Cells were treated for 48 h. DOX, doxorubicin;
LPNs-1, FA-PEG-hyd-DSPE modified PLGA nanoparticles.
5.0 than at pH 7.4. In pH 5.0 medium, LPNs-1 released
60% of the loaded DOX in 10 h. However, in pH 7.4
medium, the LPNs-1 released only 30% of the loaded
DOX in 10 h. However, the rate and amount of DOX
released from NNPs were much lower than those
released from LPNs-1. The rate and amount of DOX
released from NNPs in pH 5.0 medium were almost the
same as those in pH 6.5 medium, but were higher than
those in pH 7.4 medium. NNPs released 23% of the
loaded DOX in 10 h in pH 7.4 medium and released
about 30% of the loaded DOX in 10 h in pH 5.0 medium.
The faster release of DOX from the LPNs-1 in pH
5.0 medium resulted from the hydrazone linkage break-
ing between the DSPE and PEG, which mimicked the
drug-release behavior in endosomal/lysosomal compart-
ments of the tumor cells. The slow DOX release rate of
LPNs-1 observed in pH 7.4 medium ensured that little
0
10 20 30 40 50 60 70 80 90 100 110
Time (h)
Cumulative release profile of DOX from LPNs and NNPs in different pH
medium in vitro. (a) DOX-loaded NNPs. (b) DOX-loaded LPNs-1.
(c) DOX-loaded LPNs-2. DOX, doxorubicin; LPNs-1, FA-PEG-hyd-DSPE
modified PLGA nanoparticles; LPNs-2, PEG-hyd-DSPE modified PLGA
nanoparticles; NNPs, naked PLGA nanoparticles.
in Fig. 6. The rate and amount of DOX released from the
LPNs-1 were strongly dependent on the pH of the
medium. LPNs-1 showed much faster DOX release at pH
Copyright © Lippincott Williams & Wilkins. Unauthorized reproduction of this article is prohibited.

760 Anti-Cancer Drugs 2014, Vol 25 No 7
Fig. 8
NNPs
Dox
LNPs-1
LNPs-2
(a)
(b)
100
100
80
60
40
20
0
80
60
40
20
0
0.6
3.0
6.0 30.0
0.6
3.0
6.0 30.0
(c)
(d)
100
80
60
40
20
0
100
80
60
40
20
0
0.6
3.0
6.0 30.0
0.6
3.0
6.0 30.0
Concentration (μg/ml)
Concentration (μg/ml)
The cytotoxicity of DOX, DOX-loaded NNPs, DOX-loaded LPNs-1, and DOX-loaded LPNs-2 on tumor cells.DOX, doxorubicin; LPNs-1, FA-PEG-hyd-
DSPE modified PLGA nanoparticles; LPNs-2, PEG-hyd-DSPE modified PLGA nanoparticles; NNPs, naked PLGA nanoparticles. (a) MDA-MB-231
cells; (b) KB cells; (c) A549 cells; (d) MDA-MB-231/ADR cells. Cells were treated for 48 h.
DOX was released from LPNs-1 during circulation in
the blood.
influence the drug-release characteristics of nanoparticles
in vitro.
At the same time, LPNs-1 showed a biphasic drug-release
pattern characterized by an initial burst release of about
30, 37, and 60% of the total loaded DOX in the LPNs-1 in
10 h in pH 7.4, 6.4, and 5.0 medium, respectively, which
was mainly because of the release of DOX entrapped in
the outer lipid layer of LPNs-1. The initial burst release
was followed by a slow, but sustained release. After 100 h,
80, 45, and 37% of the entrapped drug was found to be
released, respectively, in pH 5.0, 6.4 and 7.4 medium.
The sustained release of DOX from the LPNs-1 was
attributed to diffusion-mediated release from nanoparti-
cles and the gradual hydrolysis and corrosion of the core
of LPNs-1 [56]. The faster drug release of DOX from
NNPs in 6 h was mainly because of the release of DOX
coated in the outer layer of NNPs. After this initial burst
release, DOX was released slowly but continuously at a
linear rate, indicating diffusion-mediated release from the
NNPs [56].
Cytotoxicity of nanoparticles
The cytotoxicity of DOX-loaded LPNs-1, DOX-loaded
LPNs-2, and DOX-loaded NNPs was determined using
the MTT method. Figure 7 shows the cytotoxicity of non-
drug-loaded LPNs-1 on KB cells and MDA-MB-231 cells.
The results indicated that non-drug-loaded LPNs-1 did
not influence the cell viability, which implied that blank
LPNs-1 had none of the cytotoxicity. The cytotoxicity
of DOX-loaded nanoparticles is shown in Fig. 8. As the
DOX concentration was increased, the viability of cancer
cells was decreased. DOX-loaded LPNs-1 showed greater
toxicity on KB cells, MDA-MB-231 cells, MDA-MB-231/
ADR cells, and A549 cells than DOX-loaded NNPs and
free DOX. Meanwhile, KB cells [folate receptor (FR)
overexpression] were more sensitive to DOX-loaded
LPNs-1 compared with A549 cells (FR deficient). Free
DOX (3 mmol/l) killed 27, 37, and 24% of the A549 cells,
KB cells, and MDA-MB-231 cells, respectively. The same
dose of DOX loaded in LPNs-1 killed almost 29, 58, and
56% of the A549 cells, KB cells, and MDA-MB-231
LPNs-2 showed the same drug-release character-
istics as LPNs-1, which indicated that FA did not
Copyright © Lippincott Williams & Wilkins. Unauthorized reproduction of this article is prohibited.

PEG-detachable lipid–polymer hybrid nanoparticle Du et al. 761
Fig. 9
Confocal laser scanning microscopy (CLSM) images of KB cells and MDA-MB-231/ADR cells incubated with DOX, DOX-loaded NNPs, and
DOX-loaded LPNs-1 at 371C for 4 h. DOX, doxorubicin; LPNs-1, FA-PEG-hyd-DSPE modified PLGA nanoparticles; NNPs, naked PLGA
nanoparticles. (a) Free DOX in KB cells. (b) DOX-loaded NNPs in KB cells. (c) DOX-loaded LPNs-1 in KB cells. (d) Free DOX in MDA-MB-231/ADR
cells. (e) DOX-loaded LPNs-1 in MDA-MB-231/ADR cells. The DOX concentration was 10 mg/ml. The right column shows the left and middle
columns merged, indicating the localization of DOX in the cell.
Copyright © Lippincott Williams & Wilkins. Unauthorized reproduction of this article is prohibited.

762 Anti-Cancer Drugs 2014, Vol 25 No 7
Fig. 10
(a)
86
261
170
B
B
1.3%
B
64.5%
100.0%
100 101 102 103
Control incubation 30 min
KB cells
100 101
Free DOX incubation 30 min
KB cells
102 103
100 101 102 103
Free DOX incubation 3 h
KB cells
164
168
122
B
B
99.8%
100.0%
B
100.0%
100 101 102 103
LNPs-1 incubation 30 min
KB cells
100 101
LNPs-1 incubation 3 h
102 103
100 101 102 103
LNPs-1+FA incubation 3 h
KB cells
KB cells
(b)
55
72
70
c
c
c
1.1%
87.6%
100.0%
100
101 102 103
100
101 102 103
100
101 102 103
Control incubation 30 min
MDA-MB-231/ADR cells
Free DOX incubation 30 min
MDA-MB-231/ADR cells
Free DOX incubation 3 h
MDA-MB-231/ADR cells
131
96
c
c
97.9%
100%
100
101 102 103
100
101 102
103
LNPs-1 incubation 30 min
LNPs-1 incubation 3 h
MDA-MB-231/ADR cells
MDA-MB-231/ADR cells
Flow cytometry results of cellular uptake of free DOX and DOX-loaded LPNs-1 in 30 min and 3 h. (a) KB cells; (b) MDA-MB-231/ADR cells.
The DOX concentration was 10 mg/ml. DOX, doxorubicin; LPNs-1: FA-PEG-hyd-DSPE modified PLGA nanoparticles.
Copyright © Lippincott Williams & Wilkins. Unauthorized reproduction of this article is prohibited.

PEG-detachable lipid–polymer hybrid nanoparticle Du et al. 763
Fig. 11
cells, respectively. DOX (3 mmol/l) loaded in LPNs-2
killed almost 26, 35, and 48% of the A549 cells, KB cells,
and MDA-MB-231 cells, respectively. The above data
indicated that LPNs-1 enhanced the cytotoxicity of
DOX, and there was no significant difference in
cytotoxicity on A549 cells between LPNs-1 and LPNs-
2. The high FA receptor-expressed cancer cells were more
sensitive to DOX-loaded LPNs-1 compared with DOX-
loaded LPNs-2. This implied that the FA in LPNs-1
played an important role in cytotoxicity on high FA
receptor-expressed cancer cells.
(a)
103
103
102
101
100
B1
B2
0.0%
E1
E2
0.7%
2.6%
8.2%
102
101
100
E4
11.4%
E3
85.2%
B3
91.1%
B4
0.7%
100
101
102
103
100
101
102
103
MDA-MB-231/ADR cells were the DOX-resistant cell
line. It is interesting to discover that 6 mmol/l free DOX
killed 7% of the MDA-MB-231/ADR cells. However, the
same dose of DOX loaded in LPNs-1 and LPNs-2 killed
almost 31 and 24% of MDA-MB-231/ADR cells, respec-
tively. This indicated that DOX-loaded LPNs could kill
not only normal tumor cells but also drug-resistant tumor
cells. In addition, DOX-loaded NNPs showed lower
toxicity on A549 cells, KB cells, and MDA-MB-231/ADR
cells compared with free DOX. The above results showed
that modification of PLGA with FA-PEG-hyd-DSPE
resulted in the burst release of DOX in tumor cells,
and subsequently, considerably increased the cytotoxicity
of DOX-loaded LPNs.
Control
10 μg/ml free DOX
KB cells
KB cells
103
102
101
103
102
101
E2
E1
E2
23.3%
E1
10.1%
3.9%
4.3%
100 E3
E4
100 E3
E4
64.5%
64.8%
21.2%
7.9%
100
101
102
103
100
101
102
103
10 μg/ml LNPs-1
30 μg/ml LNPs-1
KB cells
KB cells
(b)
103
103
102
101
100
Because of its small size, nanoparticles could take
advantage of the enhanced permeability and retention
effect to enhance the retention time of loaded drugs in
tumor tissue. A new and promising strategy was to
conjugate a tumor-cell-specific ligand with nanoparticles
to form an active tumor-targeting drug-delivery system,
which could selectively recognize tumor cells. Subse-
quently, it led to a reduction in drug dose, improvement
in therapeutic efficacy, and decrease in toxicity [57]. FA
was one of the most commonly used tumor-cell-specific
ligand as FR overexpression had been identified in a wide
range of tumors such as in ovarian, endometrial, color-
ectal, or breast cancer [58]. Some researches showed
enhanced uptake of folate-conjugated nanoparticles by
cancer cells [59–61].
D1
1.7%
D2
0.0%
E1
E2
0.8%
1.9%
102
101
100
D3
97.9%
D4
0.3%
E3
86.9%
E4
10.4%
100
101
Control
102
103
100
101
102
103
30 μg/ml free DOX
MDA-MB-231/ADR cells
MDA-MB-231/ADR cells
103
102
101
100
103
102
101
100
E1
1.5%
E2
6.1%
E1
2.7%
E2
6.9%
E4
51.8%
E4
22.7%
E3
40.5%
E3
67.7%
In-vitro cellular uptake of NPs
The main anticancer action of DOX was to inhibit
topoisomerase II and cause DNA damage. Chromosomal
DNA was the main action target of DOX. Thus, the
subcellular distribution of DOX was evaluated by confocal
laser scanning microscopy. After KB cells were treated
with free DOX for 4 h, DOX was predominantly
accumulated in the nucleus (Fig. 9a). When MDA-MB-
231/ADR cells were treated with free DOX for 4 h, DOX
was predominantly accumulated in the cytoplasma
(Fig. 9d). The cellular uptake of DOX-loaded LPNs-1
in KB cells and MDA-MB-231/ADR cells is, respectively,
shown in Fig. 9c and e. When KB cells and MDA-MB-231/
ADR cells were incubated with DOX-loaded LPNs-1, a
100
10 μg/ml LNPs-1
MDA-MB-231/ADR cells
101
102
103
100
30 μg/ml LNPs-1
MDA-MB-231/ADR cells
101
102
103
Apoptosis of KB cells (a) and MDA-MB-231/ADR cells (b) induced
by different concentrations of DOX and DOX-loaded LPNs-1.
DOX, doxorubicin; LPNs-1: FA-PEG-hyd-DSPE modified PLGA
nanoparticles. KB cells and MDA-MB-231/ADR cells were treated with
10 mmol/l free DOX or 10, 30 mmol/l DOX-loaded LPNs-1 for 24 h.
Cells were harvested by trypsinization and centrifugation, and then
analyzed in a Becton Dickinson FACScan (excitation at 488 nm)
equipped with Cell Quest software (Becton Dickinson
Immunocytometry Systems, San Diego, California, USA) after staining
with annexin V-FITC and propidium iodide.
Copyright © Lippincott Williams & Wilkins. Unauthorized reproduction of this article is prohibited.

764 Anti-Cancer Drugs 2014, Vol 25 No 7
large amount of DOX was distributed in the nucleus.
This was expected because of the dissociation of
FA-PEG-hyd-DSPE on the surface of LPNs-1 in endo-
lysosomes, subsequently resulting in the burst release of
DOX and a similar pattern of cellular distribution with
free DOX. However, a small amount of DOX was
distributed in the nucleus after DOX-loaded NNPs were
incubated with KB cells (Fig. 9b).
Conclusion
The above experiment implied that modification of PLGA
nanoparticles with FA-PEG-hyd-DSPE enhanced the drug
loading. Compared with NNPs, LPNs-1 significantly
increased the intranuclear distribution of DOX both in
normal tumor cells and in DOX-resistant tumor cells.
Therefore, it was concluded that modification of PLGA
nanoparticles with FA-PEG-hyd-DSPE could considerably
enhance the drug-delivery efficiency and it had potential in
delivering chemotherapeutic agents to treat cancer.
The cellular uptake of free DOX and DOX-loaded LPNs-
1 was further investigated semiquantitatively in KB cells
and MDA-MB-231/ADR cells using flow cytometry. The
results are shown in Fig. 10a and b. The cellular uptake of
free DOX and DOX-loaded LPNs-1 in KB cells and
MDA-MB-231/ADR cells increased in a time-dependent
manner. The intracellular uptake of DOX-loaded LPNs-1
in KB cells and MDA-MB-231/ADR cells was greater than
that of free DOX. When KB cells were incubated with
exogenous folate and DOX-loaded LPNs-1, the uptake
efficiency of DOX-loaded LPNs-1 was attenuated
obviously. These results clearly indicated that cellular
uptake of DOX-loaded LPNs-1 on KB cells was facilitated
by receptor-mediated endocytosis.
Acknowledgements
This work was supported by the Shaanxi Science
and Technology Innovation Project (2012KTCL03-18) and
the Shaanxi Natural Science Foundation (2013JQ4026).
Conflicts of interest
There are no conflicts of interest.
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