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Combined Delivery of Temozolomide and siPLK1 Using Targeted Nanoparticles to Enhance Temozolomide Sensitivity in Glioma

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Introduction: Temozolomide (TMZ) is the first-line chemotherapeutic option to treat glioma; however, its efficacy and clinical application are limited by its drug resistance properties. Polo-like kinase 1 (PLK1)-targeted therapy causes G2/M arrest and increases the sensitivity of glioma to TMZ. Therefore, to limit TMZ resistance in glioma, an angiopep-2 (A2)-modified polymeric micelle (A2PEC) embedded with TMZ and a small interfering RNA (siRNA) targeting PLK1 (siPLK1) was developed (TMZ-A2PEC/siPLK). Materials and methods: TMZ was encapsulated by A2-PEG-PEI-PCL (A2PEC) through the hydrophobic interaction, and siPLK1 was complexed with the TMZ-A2PEC through electrostatic interaction. Then, an angiopep-2 (A2) modified polymeric micelle (A2PEC) embedding TMZ and siRNA targeting polo-like kinase 1 (siPLK1) was developed (TMZ-A2PEC/siPLK). Results: In vitro experiments indicated that TMZ-A2PEC/siPLK effectively enhanced the cellular uptake of TMZ and siPLK1 and resulted in significant cell apoptosis and cytotoxicity of glioma cells. In vivo experiments showed that glioma growth was inhibited, and the survival time of the animals was prolonged remarkably after TMZ-A2PEC/siPLK1 was injected via their tail vein. Discussion: The results demonstrate that the combination of TMZ and siPLK1 in A2PEC could enhance the efficacy of TMZ in treating glioma.
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ORIGINAL RESEARCH
Combined Delivery of Temozolomide and siPLK1
Using Targeted Nanoparticles to Enhance
Temozolomide Sensitivity in Glioma
This article was published in the following Dove Press journal:
International Journal of Nanomedicine
Hui Shi
1,2,
*
Shuo Sun
1,
*
Haoyue Xu
3,
*
Zongren Zhao
3
Zhengzhong Han
3
Jun Jia
3
Dongmei Wu
4
Jun Lu
4
Hongmei Liu
3,5
Rutong Yu
1,3,5
1
Clinical Medical College, Nanjing
Medical University, Nanjing, Peoples
Republic of China;
2
The Second Peoples
Hospital of Lianyungang, Lianyungang,
Peoples Republic of China;
3
Institute of
Nervous System Diseases, Xuzhou
Medical University, Xuzhou, Peoples
Republic of China;
4
Key Laboratory for
Biotechnology on Medicinal Plants of
Jiangsu Province, School of Life Science,
Jiangsu Normal University, Xuzhou,
Jiangsu, Peoples Republic of China;
5
Department of Neurosurgery, Afliated
Hospital of Xuzhou Medical University,
Xuzhou, Peoples Republic of China
*These authors contributed equally to
this work
Introduction: Temozolomide (TMZ) is the rst-line chemotherapeutic option to treat
glioma; however, its efcacy and clinical application are limited by its drug resistance
properties. Polo-like kinase 1 (PLK1)-targeted therapy causes G2/M arrest and increases
the sensitivity of glioma to TMZ. Therefore, to limit TMZ resistance in glioma, an angiopep-
2 (A2)-modied polymeric micelle (A2PEC) embedded with TMZ and a small interfering
RNA (siRNA) targeting PLK1 (siPLK1) was developed (TMZ-A2PEC/siPLK).
Materials and Methods: TMZ was encapsulated by A2-PEG-PEI-PCL (A2PEC) through
the hydrophobic interaction, and siPLK1 was complexed with the TMZ-A2PEC through
electrostatic interaction. Then, an angiopep-2 (A2) modied polymeric micelle (A2PEC)
embedding TMZ and siRNA targeting polo-like kinase 1 (siPLK1) was developed (TMZ-
A2PEC/siPLK).
Results: In vitro experiments indicated that TMZ-A2PEC/siPLK effectively enhanced the
cellular uptake of TMZ and siPLK1 and resulted in signicant cell apoptosis and cytotoxicity
of glioma cells. In vivo experiments showed that glioma growth was inhibited, and the
survival time of the animals was prolonged remarkably after TMZ-A2PEC/siPLK1 was
injected via their tail vein.
Discussion: The results demonstrate that the combination of TMZ and siPLK1 in A2PEC
could enhance the efcacy of TMZ in treating glioma.
Keywords: co-delivery, polymeric micelle, siPLK1, TMZ, drug resistance, glioma
Introduction
Glioma is the most common and aggressive primary brain tumor in the central
nervous system (CNS), accounting for approximately 40% of brain tumors.
1
The
standard treatments for glioma include surgery, radiation therapy, and
chemotherapy.
2
However, the presence of the blood-brain barrier (BBB), genetic
heterogeneity, and the ability to invade and/or inltrate adjacent tissues of glioma
cells,
3,4
has resulted in poor therapeutic effects in glioma.
5,6
The median survival
time is 14.6 months and the ve-year survival rates is less than 10%.
7,8
Therefore, it
is important to apply chemotherapy drugs to patients with glioma after surgery and
to prevent postoperative recurrence.
911
TMZ is a second-generation imidazotetrazine derivative and is a standard che-
motherapeutic drug for gliomas.
2
The cytotoxic effect of TMZ acts mainly through
the methylation of DNA guanine and the formation of O-6-methylguanine
(O
6
meG).
12,13
In the process of DNA replication, O
6
meG mismatches thymine,
Correspondence: Rutong Yu; Hongmei
Liu
Tel +86 13182310079; +86 17716228111
Email yu.rutong@163.com;
liuhongmei816@sina.com
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resulting in ineffective mismatch repair and persistent G2
checkpoint arrest, eventually leading to apoptosis of tumor
cells.
1315
However, the effectiveness of TMZ in clinical
treatment is limited because of the following problems: 1)
Most gliomas gradually develop drug resistance to TMZ
during chemotherapy;
5,14
2) the indiscriminate attack on
DNA has been shown to cause damage to hematopoietic
stem cells;
13
3) TMZ is difcult to dissolve and is suscep-
tible to rapid hydrolysis under physiological conditions;
1
and 4) the obstruction of the BBB further limits its anti-
glioma efcacy.
16
In the last few years, many pre-clinical
studies have used nanoparticles (NPs) as vehicles to deli-
ver TMZ effectively to gliomas, and have resolved the last
three questions above.
1,13,16
However, drug resistance to
TMZ has not been improved effectively.
17
Therefore, there
is an urgent need to nd a new therapeutic strategies to
limit the drug resistance to TMZ in glioma.
Polo-like kinase 1 (PLK1) is a serine/threonine protein
kinase that is involved in spindle formation and chromo-
some segregation during mitosis, and plays a key regula-
tory role in the cell cycle.
18,19
Clinical pathological data
showed that the expression of PLK1 in glioma tissues was
signicantly higher than that in normal brain tissues.
20
Inhibition of PLK1 can cause cell cycle arrest and increase
apoptosis in glioma cells.
21,22
Besides, studies have shown
that small-molecule kinase inhibitors of PLK1 increase the
sensitivity of tumor cells to chemotherapeutics.
2326
Koncar et al reported that a combination of TMZ and
a PLK1 inhibitor, BI2536, signicantly suppressed glioma
tumor growth.
27
Liu et al found that a PLK1 inhibitor
combined with TMZ efciently induced G2/M arrest and
suppressed cell proliferation and sphere formation in
glioma cells.
28
These ndings suggested that
a combination treatment comprising TMZ and a PLK1
inhibitor could reverse TMZ resistance efciently in
glioma treatment. However, the clinical application of
small-molecule kinase inhibitors is greatly affected by off-
target and toxic effects.
29,30
Knockdown of PLK1 using
a small interfering RNA (siRNA) has become a new ther-
apeutic strategy,
3134
and the US Food and Drug
Administration (FDA) has approved the application of
the siRNA therapy in clinical practice.
35
Our previous
study successfully delivered siPLK1 into glioma cells
using hypoxia-responsive ionizable liposomes, which
inhibited the growth of glioma cells efciently, both
in vitro and in vivo.
36
However, to date, there has been
no study on the combination treatment of TMZ and
siPLK1 using a targeted NP delivery system.
In the present study, we constructed an NP drug deliv-
ery system to co-deliver TMZ and siPLK1 into glioma
cells, with the hope of enhancing TMZ sensitivity and
apoptosis in glioma treatment. We used the angiopep-2
(A2) to modify polymeric micelles, because A2-modied
polymers can penetrate the BBB through receptor-
mediated transport and accumulate in the brain in large
quantities. Polymers modied by A2 to deliver drugs
through the BBB have achieved certain effects in treating
CNS diseases and malignant gliomas.
37
TMZ was encap-
sulated by A2-poly(ethyleneglycol) (PEG)-poly(ethyleni-
mine) (PEI)-poly(Ɛ-caprolactone) (PCL) (A2PEC)
micelles through hydrophobic interactions. Then, siPLK1
was complexed with the TMZ-A2PEC micelles through
electrostatic interaction. TMZ-A2PEC/siPLK1 could pro-
mote the penetration of siPLK1 across the BBB and pro-
tect siPLK1 from degradation. In addition, the combined
delivery of TMZ and siPLK1 enhanced the sensitivity of
glioma cells to TMZ, consequently increasing its anti-
tumor activity both in vitro and in vivo.
Materials and Methods
Materials
Ortho-pyridyl disulde (OPSS)-PEG-succinimidyl valeric
acid (SVA) (OPSS-PEG-SVA) was obtained from Laysan
Bio, Inc (Tower Drive, Arab, AL, USA). PCL
5000
-PEI
2000
was purchased from Xian Ruixi Biological Technology
Co., Ltd (Xian, China). TMZ and D-Luciferin potassium
salt were obtained from Dalian Meilun Biotech Co., Ltd
(Dalian, Peoples Republic of China). 4,6-diamidino-
2-phenylindole dihydrochloride (DAPI) was purchased
from Sigma-Aldrich (St. Louis, MO, USA). Annexin
V-uorescein isothiocyanate (FITC)/propidium iodide
(PI) was obtained from Nanjing KeyGEN BioTECH Co.
Ltd (Nanjing, Peoples Republic of China).
Hypoxyprobe
TM
-1 Plus Kit was purchased from
Hypoxyprobe, Inc. (Burlington, MA, USA). Angiopep-2
(TFFYGGSRGKRNNF KTEEY) was purchased from GL
Biochem Ltd (Shanghai, China). 3-(4,5-Dimethylthiazol-
2-yl)-2,5-diphenyltetrazolium bromide (MTT) was pur-
chased from Beijing Zhongshuo Pharmaceutical
Technology Development Co., Ltd. LysoTrackerred
was bought from Invitrogen (Carlsbad, CA, USA). PLK1
(208G4) Rabbit monoclonal antibodies (mAbs) were
obtained from Cell Signaling Technology Co., Ltd
(Danvers, MA, USA). Beta-actin mAbs were bought
from Proteintech Antibodies People Trust (Chicago, IL,
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USA). DiOC18
7
(DiR) was brought from Suzhou
Biosyntech Co., Ltd (Suzhou, Peoples Republic of
China). FAM-labeled siRNA (FAM-siRNA), negative con-
trol siRNA with a scrambled sequence (nonsense, anti-
sense strand, 5-ACGUGACACGUUCGGAGAAdTdT
-3), and siRNA targeting PLK1 mRNA (siPLK1, anti-
sense strand, 5-AGAUCACUCUCCUCAACUAUU-3)
were purchased from GenePharma Co. Ltd. (Shanghai,
Peoples Republic of China).
Methods
Nanoparticle Preparation
OPSS-PEG-SVA and A2 (molar ratio: 10:1) were dissolved
in dimethyl sulfoxide (DMSO, Sigma, Neustadt, Germany).
The reaction mixture was stirred gently at room temperature
for 36 h, ltered, dialyzed against deionized water (mole-
cular weight cut off: 1 kDa), and lyophilized to obtain A2-
modied OPSS-PEG-SVA (A2-OPSS-PEG-SVA).
A2-OPSS-PEG-SVA (1 mg) and PCL
5000
-PEI
2000
(2 mg) were completely dissolved in acetone and vortexed
vigorously for 2 min at room temperature. The mixture
was dripped into pure water and stirred with a magnetic
stirrer for 30 min and puried by membrane dialysis
(molecular weight cut off: 8000 Da) against water for 24
h. This process formed A2-PEG-PEI-PCL, which was
abbreviated as A2PEC. TMZ-A2PEC was prepared using
A2-OPSS-PEG-SVA (1 mg), PCL
5000
-PEI
2000
(2 mg), as
TMZ (2 mg) according to the above method.
A predetermined (eg, 0.1 µg) amount of siPLK-1 A or
negative control siRNA (NCsiRNA) was mixed with
a certain amount of TMZ-A2PEC micelle solution. The
mixture was vortexed for 15 s and then left for 30 min at
room temperature. By this means, a series of siRNA and
TMZ-loaded nanocomplexes (TMZ-A2PEC/siPLK1 and
TMZ-A2PEC/NCsiRNA) were formed according to differ-
ent nitrogen/phosphate (N/P) ratios. Nanocomplexes with-
out TMZ (A2PEC/siPLK1) or containing FAM-labeled
siRNA (A2PEC/FAM-siRNA) were prepared in the
same way.
Characterization of NPs
The nanocomplexes were freshly prepared. The mean size
and zeta potential were determined using a Malvern
Zetasizer Nano instrument (Malvern Panalytical,
Malvern, UK). For each sample, data obtained from three
measurements were averaged to yield the mean size and
zeta potential. The morphology of the NPs was detected
using transmission electron microscopy (TEM, FEI Tecnai
G2 T12; Thermo Fisher Scientic,Waltham, MA, USA).
Gel Retardation Assay
The binding ability of the siRNA to the TMZ-loaded
micelles was estimated using agarose gel electrophoresis.
Gel electrophoresis was performed using 2% (w/v) agar-
ose gel in TAE buffer with 0.5 µg mL
1
of ethidium
bromide (EtBr). Complexation of siRNAs at various N/P
ratios 0, 0.3, 0.5, 1, 1.5, and 2 were prepared for electro-
phoresis. Samples were run at 110 V for 15 min. The result
was visualized using the UV exposure.
Drug Loading Efciency
The TMZ-loading efciency, dened as the weight percen-
tage of TMZ in the micelle, was determined using a high
performance liquid chromatography (HPLC) system
equipped with a column (C18, 5 µm, 200 mm, Dikma
Technologies Inc., Foothill Ranch, CA, USA). First, the
TMZ-loaded micelle was dissolved in acetone, and
a calibration curve was obtained using acetone solutions
with different TMZ concentrations. Then, the TMZ-loaded
micelle solutions were lyophilized, weighed, and redis-
solved in acetone. The mixture was centrifuged for 30
min using a table-top centrifuge (Micro 17R, HANIL
SME, Co., Ltd., Seoul, Korea). After centrifugation, the
upper phase of the mixture was measured spectrophotome-
trically at 328 nm. The loading efciency and loading
content of TMZ were calculated using the following
formulas:
Loadingefficiency %ðÞ¼WzWu
ðÞ=Wz100%
Loadingcontent %
ðÞ
¼WzWu
ðÞ
=Wt100%
W
z
: the weight of the initial TMZ (mg); W
u
: the weight of
TMZ detected in the upper phase after centrifugation (mg);
W
t
: the total weight of lyophilized NPs (mg).
Cell Culture
The glioma cell lines LN-229, T98G, and U87 cell lines
were obtained from the Shanghai Cell Bank, Type Culture
Collection Committee, Chinese Academy of Sciences.
U87-GFP-Luci cells were transformed with the luciferase
gene. U87R, a TMZ-resistant clone of U87, was estab-
lished by treating U87 cells with a low dose of TMZ in
DMEM for 3 weeks. All cells were routinely cultured in
Dulbeccos modied Eagles medium (DMEM) (Gibco,
Carlsbad, CA, USA) containing 10% fetal bovine serum
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(heat-inactivated) (FBS, Gibco). The cells were cultured at
37 °C in a humidied atmosphere of 5% CO
2
.
Cellular Uptake
FAM-siRNA was used as uorescent probe of the NPs to
assess their cellular uptake. Free FAM-siRNA, PEC/FAM-
siRNA,and A2PEC/FAM-siRNA were prepared. U87R
cells were seeded in 12-well culture plates at a density of
5×10
4
and incubated at 37 °C in a humidied atmosphere
of 5% CO
2
with DMEM containing 10% FBS (heat-
inactivated) for 24 h. The old medium was then replaced
with serum-free medium. Free FAM-siRNA, PEC/FAM-
siRNA, and A2PEC/FAM-siRNA at a dose of FAM-
siRNA 1 µg mL
1
were added, and after the cells were
incubated for 4 h, they were trypsinized, centrifuged at
2000 rpm for 5 min, washed three times with cold phos-
phate-buffered saline (PBS), and resuspended in 500 µL of
PBS. Later, the cells were measured using a BD
FACSCalibur Flow Cytometer (BD Biosciences, San
Jose, CA, USA).
Confocal Laser Scan Microscopy
To assess the cellular uptake and endosomal escape of the
NPs, confocal laser scanning microscopy (CLSM) was
used. U87R cells were seeded in a 35-mm glass bottom
culture dish at a density of 5 × 10
4
and incubated at 37 °C
in a humidied atmosphere of 5% CO
2
for 24 h. The old
medium was then replaced by serum-free medium.
A2PEC/FAM-siRNA at a dose of FAM-siRNA of 1
µg mL
1
was added and the cells were incubated for 2
h and 4 h, respectively. Later, the medium was removed,
the cells were stained with Hoechst 33342 (1: 1,000) for
10 min, and then washed three times with PBS. The cells
were then stained with LysoTracker Red (1: 10,000). The
CLSM imaging was conducted using an FV10i laser scan-
ning microscope (Olympus, Tokyo Japan). Hoechst 33342,
FAM-siRNA, and LysoTracker Red were excited at 352,
480, and 577 nm, respectively.
In vitro Cytotoxicity Assay
An MTT assay was performed to assess the cytotoxicity of
the nanocomplexes. LN-229 and U87R cells were seeded
at a density of 5 × 10
3
cells/well in 96-well plates and
incubated for 12 h. There were six dosage regimens: 1)
PBS; 2) free TMZ; 3) free siPLK1; 4) A2PEC micelles
complexed with siPLK1 (A2PEC/siPLK1); 5) A2PEC
micelles loaded with TMZ and NCsiRNA (TMZ-A2PEC
/NCsiRNA); and 6) A2PEC micelles loaded with TMZ
and siPLK1 (TMZ-A2PEC/siPLK1). The amount of
applied siRNA per well in cell culture was set to 1
µg mL
1
, the amount of TMZ per well was 15 µg mL
1
.
The cells were then incubated for 48 h before 20 μmL of
MTT solution (5 mg/mL) in PBS buffer was added to each
well and the cells were cultured for another 4h. After the
removal of the MTT medium, 150 µL of DMSO was
added to each well. The optical density (OD) at 570 nm
was measured using spectrophotometric analysis.
Cell Cycle Analysis
LN-229 and U87R cells were seeded at a density of 5 × 10
4
cells/well in 12-well plate and incubated for 12 h. The cells
were then treated with PBS, free TMZ, free siPLK1,
A2PEC/siPLK1, TMZ-A2PEC/NCsiRNA, or TMZ-A2PEC
/siPLK1. Treatments were given at doses of 15 µg mL
1
TMZ and 1 µg mL
1
siRNA. After the cells were incubated
for 48 h, these cells were harvested and washed three times
with PBS. Then cells were centrifugated (1,000 rpm, 10
min), the supernatant was discarded, and the cell pellet
was xed in 70% ethanol at 4 °C for 24 h. Before analysis,
the cells were washed once in PBS, stained with 500 µL PI/
RNase for 15 min in the dark at room temperature. Analyses
were performed using a BD FACSCalibur Flow Cytometer.
Cell Cycle distributions were calculated using BD FACS
Diva software (Verity Software House).
Cell Apoptosis Study
LN-229 and U87R cells were seeded at a density of 1 ×
10
5
cells/well in 6-well plate and cultured at 37 °C in a 5%
CO
2
for 24 h. The cells were then treated with different
micelle formulations (PBS, free TMZ, free siPLK1,
A2PEC/siPLK1, TMZ-A2PEC/NCsiRNA, and TMZ-
A2PEC/siPLK1). Treatments were given at doses of 15
µg mL
1
TMZ and 1 µg mL
1
siRNA. After the cells were
incubated for 48 h, they were trypsinized, collected,
washed three times with PBS, and resuspended in 500
μL of binding buffer. Then, 5 μL of Annexin V-FITC
and 5 μL PI were added, and cultured with the cells for
15 min in the dark. Finally, cell apoptosis was evaluated
using a ow cytometer.
Quantitative Real-Time Reverse
Transcription Polymerase Chain Reaction
Analysis
The expression levels of PLK1 mRNA in U87R cells of
different groups were evaluated using quantitative real-time
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reverse transcription polymerase chain reaction (qRT-PCR).
Total RNA was extracted from cell using the TRIzol reagent
(TIANGEN, Beijing, Peoples Republic of China) according
to the manufacturers instructions. The extracted RNA was
digested with DNase I to remove possible DNA contamina-
tion. First strand cDNAwas synthesized with the RNA which
had been puried again using the TRIzol reagent, using
a reverse transcription kit. Finally, the quantitative analysis
of the cDNA was performed using qPCR in an ABI 750
machine (ABI, Foster City, CA, USA). The GAPDH (encod-
ing glyceraldehyde-3-phosphate dehydrogenase) mRNA
level was measured as an internal normalization standard.
All primers were synthesized by Sangon Biotech (Shanghai,
Peoples Republic of China).
Western Blotting Analysis
The PLK1 protein was detected in U87R cells using
Western blotting. U87R cells (2 × 10
5
) were seeded in
a 6-well culture plate and incubated at 37 °C in a 5% CO
2
for 24 h. The cells were then treated with PBS, free TMZ,
free siPLK1, A2PEC/siPLK1, TMZ-A2PEC/NCsiRNA, or
TMZ-A2PEC/siPLK1 at doses of 15 µg mL
1
TMZ and 1
µg mL
1
siRNA for 24 h. The cells were then harvested,
washed twice with cold PBS, and lysed. The lysis buffer
was centrifuged (12,000 rpm, 10 min) to remove impurities.
Quantities and equilibriums of the extracted protein sam-
ples were measured using a BCA kit (Beyotime, Shanghai,
Peoples Republic of China). The proteins were then sepa-
rated using SDS-PAGE and transferred onto a nitrocellulose
membrane using SemiDry blot apparatus (Invitrogen). The
membrane was then incubated with primary antibodies
overnight and secondary antibodies for 1.5 h. Finally, the
membranes were exposed to x-ray lms in a dark room. The
band densities were quantied using the Image J Software
(NIH. Bethesda, MD, USA. The expression of
O-6-Methylguanine-DNA Methyltransferase (MGMT) in
T98G, LN-229, U87, and U87R cells were evaluated
using the same method.
Glioma Model
BALB/c nude mice (male, 5 weeks old) were purchased
from Beijing Vital River Laboratory Animal Technology
Co., Ltd. (Beijing, China). All animals received care fol-
lowing the guidelines for the Guide for the Care and Use
of Laboratory Animals. All experiment procedures were
approved by Xuzhou Medical University of China Animal
Care and Use Committee (license no. SYXK 20020038;
Jiangsu, china). The glioma model was constructed by
intracranial injection (striatum, 1.8 mm right lateral to
the bregma and 3 mm in depth) of 8 × 10
5
U87-luci
cells suspended in 7 µL of L15 medium into the male
nude mice. After 10 days of xenograft glioma, the inten-
sity of luciferase uorescence was measured using an
in vivo imaging system (Caliper, Princeton, NJ, USA) to
conrm successful construction of the nude mice glioma
model. Thereafter, the nude mice were randomly divided
into six groups (n = 6) and housed in a controlled-
temperature room with highly sterilized standard feed
and water.
Biodistribution Studies
The biodistribution studies were determined after the nude
mice glioma model was proven to be constructed success-
fully. PBS, free DiR, and A2PEC/DiR (the dose of DiR
was 1 mg kg
1
) were injected through the tail vein, respec-
tively. The nude mice were sacriced 4 h after adminis-
tration, and the glioma model brains and their organs
(heart, liver, spleen, lung and kidney) were collected.
The in vivo imaging system (Caliper) was used to observe
the intensity of DiR staining.
In vivo Antitumor Efcacy
After U87 cells were implanted for 10 days, the mice were
divided into six groups (n = 6). Each group received an i.v.
injection via the tail vein with PBS, free TMZ, free
siPLK1, A2PEC/siPLK1, TMZ-A2PEC/NCsiRNA, or
TMZ-A2PEC/siPLK1, respectively. The bioluminescence
intensity which was used to measure the size of glioma
was 9×10
7
µw cm
2
when the therapy was initiated. All
groups were treated on day 12, day 14, day 16, and day 18
at doses of 10 mg kg
1
TMZ and 1 mg kg
1
siRNA. The
mice were imaged again using the in vivo imaging system
(Caliper) on day 20 and day 30. During the in vivo experi-
ment, the survival time of each group was observed, and
the weight of the mice was also recorded every two days.
Organ Safety Evaluation
The U87-Luci tumor-bearing mice received injections of
PBS, free TMZ, free siPLK1, A2PEC/siPLK1, TMZ-
A2PEC/NCsiRNA, and TMZ-A2PEC/siPLK1 at doses of
10 mg kg
1
TMZ and 1 mg kg
1
siRNA on days 12, 14,
16, and 18. One day after the last treatment, three mice
from each group were sacriced, their organs (heart, liver,
spleen, lung and kidney) were collected and sections of the
organs were stained with hematoxylin and eosin (H&E)
for histological examination.
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Statistical Analysis
Statistical analysis of the data was performed using analy-
sis of variance (ANOVA) and Studentst-tests, Pvalues <
0.05 was considered statistically signicant. The results
were expressed as mean and mean ± SD in the gures
(*P< 0.05, **P< 0.01, ***P< 0.001).
Results
Preparation and Characterization of
TMZ-A2PEC/siPLK1
TMZ-A2PEC/siPLK1 was synthesized as outlined in Figure 1.
TMZ was encapsulated into the micelle core through hydro-
phobic interactions to form TMZ-A2PEC. The naked siPLK1
was easily complexed with the TMZ-A2PEC via electrostatic
interaction to form TMZ-A2PEC/siPLK1. Agarose gel elec-
trophoresis showed that siPLK1 was completely adsorbed by
TMZ-A2PEC when the N/P ratio was 1:1 (Figure 2A). The
TEM image showed that TMZ-A2PEC/siPLK1 had a regular
spherical shape (Figure 2B). The average particle sizes of
A2PEC, TMZ-A2PEC, and TMZ-A2PEC/siPLK1 were
72.94 nm, 76.56 nm, and 82.22 nm, respectively (Figure 2C
[a-c]). The zeta potentials of A2PEC, TMZ-A2PEC, and
TMZ-A2PEC/siPLK1 were 26.00 mV, 26.60 mV, and 18.80
mV, respectively (Figure 2D [a-c]). The TMZ loading ef-
ciency and loading content of TMZ-A2PEC/siPLK1 were
91.69% and 9.17%, respectively.
Cellular Uptake and Endosomal Escape
The cellular uptake of the A2PEC/FAM-siRNA was investi-
gated after the U87R cells were treated with PBS, free FAM-
siRNA, PEC/FAM-siRNA, and A2PEC/FAM-siRNA
(Figure 3A). The ow cytometry results indicated that the
cells incubated with A2PEC/FAM-siRNA showed the stron-
gest FAM-siRNA uorescence among all the groups. This
result indicated that the A2 modication enhanced the cellu-
lar uptake of A2PEC/FAM-siRNA. In addition, to estimate
the effect of endosome escape of the A2PEC/FAM-siRNA in
glioma cells, CLSM was used. Cellular endosome escape
was monitored after the glioma cells were incubated with
A2PEC/FAM-siRNAfor2and4h(Figure 3B). At 2 h, the
green FAM-siRNA overlapped with the red endosomes,
indicating the uptake of A2PEC/FAM-siRNA into the cyto-
sol. However, at 4 h, the green FAM-siRNA deviated from
red endosomes, indicating that the A2PEC/FAM-siRNA had
escaped from the endosomes.
DNA damage
Sensitize
TMZ
RISC
G2/M arrest
Apoptosis
Nucleus
G2/M G1
S
siPLK1
Self-assembly & electrostatic interaction
TMZ OPSS-PEG-SVA
PEI-PCLAngiopep-2
AB
Figure 1 Schematic representation of the TMZ-A2PEC/siPLK1 drug formation and delivery system. (A) The main components and formation of TMZ-A2PEC/siPLK1. (B)
After intravenous (i.v.) injection, the TMZ-A2PEC/siPLK1 preferentially accumulate in glioma by receptor mediated transcytosis (RMT) strategy and internalize through
endocytosis, then TMZ-A2PEC/siPLK1 escape from the endosomal and release the TMZ and siPLK1, the siPLK1 silence the PLK1 gene and cause the G2/M arrest, thereby
enhance the efcacy of TMZ in treating glioma.
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Cell Cycle Arrest
We treated U87R and LN-229 cells with PBS, free siPLK1,
A2PEC/siPLK1, free TMZ, TMZ-A2PEC/NCsiRNA, and
TMZ-A2PEC/siPLK1 for 48 h, separately, and observed
the cell cycle arrest using ow cytometry (Figure 3CE).
The percentages of U87R cells in the G2/M were 6.82%
(PBS), 10.81% (free siPLK1), 29.96% (A2PEC/siPLK1),
18.7% (free TMZ), 24.14% (TMZ-A2PEC/NCsiRNA), and
40.33% (TMZ-A2PEC/siPLK1). However, in the LN-229
cells, the percentages were 8.47% (PBS), 14.20% (free
siPLK1), 32.64% (A2PEC/siPLK1), 19.38% (free TMZ),
25.20% (TMZ-A2PEC/NCsiRNA), and 43.49% (TMZ-
A2PEC/siPLK1). These results showed that both siPLK1
and TMZ alone could induce G2/M arrest, and when the
cells were treated with the TMZ-A2PEC/siPLK1, the per-
centage of cells in the G2/M phase was the highest among
the groups. These ndings indicated that siPLK1 could
enhance sensitivity to TMZ by inducing cell cycle arrest,
and also explained why more cells could enter the apoptosis
phase after incubation with TMZ-A2PEC/siPLK1.
Cell Apoptosis
Annexin V-FITC/PI double-staining assay was used to
quantitatively evaluate cell apoptosis by ow cytometry
(Figure 4). The control group without treatment showed an
extremely low apoptosis rate of 0.70%. In contrast, the
cells incubated with the TMZ-A2PEC/siPLK1 showed the
highest apoptosis rate of 20.91%. The free siPLK1,
A2PEC/siPLK1, free TMZ, and TMZ-A2PEC/NCsiRNA
treatments resulted in cell apoptosis rates of only 1.53%,
3.31%, 8.85%, and 10.55%, respectively. These results
further demonstrated that the combination of siPLK1 and
TMZ could effectively induce glioma cell apoptosis.
Gene Silencing Ability and Establishment
of TMZ-Resistant Cell Line U87R
The transcription and translation of PLK1 were evaluated
using qRT-PCR and Western blotting, respectively. QRT-
PCR showed that treatment with TMZ-A2PEC/siPLK1
and A2PEC/siPLK1 signicantly silenced PLK1 gene
expression in glioma cells (Figure 5E), leading to 61%
AB
C
D
abc
abc
Figure 2 Preparation and characterization of TMZ-A2PEC/siPLK1. (A) Gel retardation assay of binding capacity of siPLK1 at various N/P ratios. (B) TEM image of TMZ-
A2PEC/siPLK1. (C) The particle size distribution of A2PEC (a), TMZ-A2PEC (b) and TMZ-A2PEC/siPLK1 (c). (D) The zeta potential of A2PEC (a), TMZ-A2PEC (b) and
TMZ-A2PEC/siPLK1 (c).
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and 60% knockdown of PLK1 mRNA, respectively.
Meanwhile, incubation of cells with free siPLK1 only
resulted in 21% knockdown of PLK1 mRNA. However,
the cells treated with PBS, free TMZ, and TMZ-A2PEC
/NCsiRNA showed no knockdown efciency. In the
Western blotting experiment (Figure 5A), the TMZ-
A2PEC/siPLK1 and A2PEC/siPLK1 groups exhibited sig-
nicantly decreased PLK1 protein levels (by 68% and
50%) compared with that induced in the free siPLK1
group (30%); the control, free TMZ, and TMZ-A2PEC
/NCsiRNA groups showed no effect on the PLK1 protein
expression (Figure 5C). These results were consistent with
the qRT-PCR data, and both indicated that TMZ-A2PEC
/siPLK1 and A2PEC/siPLK1 exerted a strong gene silen-
cing effect on PLK1.
It has been reported that the glioma cell lines with high
MGMT levels demonstrate drug resistance in the presence of
TMZ.
38
To demonstrate that the U87R cells were resistant to
the TMZ, the MGMT protein level was evaluated using
Western blotting (Figure 5B). The MGMT protein level in
U87R cells (59%) was signicantly higher than that in U87
(8%) and LN-229 cells (11%), which were relatively sensitive
Figure 3 Cellular uptake and endosomal escape of A2PEC/FAM-siRNA in U87R cells. G2/M cell cycle arrest in U87R and LN-229 cells after treatment. (A) Flow cytometric
analysis of U87R cells incubated with PBS, free FAM-siRNA, PEC/FAM-siRNA and A2PEC/FAM-siRNA for 4 h. (B) Endosomal escape of A2PEC/FAM-siRNA in U87R cells.
The nucleus was stained by Hoechst 33342, endosome/lysosome was stained by LysoTraker Red, and FAM-siRNA emitted green uorescence by itself (scale bar = 10 µm).
(C) Cell cycle analyses of U87R and LN-229 cells were conducted after treatment with PBS, free siPLK1, A2PEC/siPLK1, free TMZ, TMZ-A2PEC/NCsiRNA and TMZ-
A2PEC/siPLK1 for 48 h. (Dand E) Percentages of cells in each cell cycle stage, the data were reported as the averages of triplicate experiments.
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Figure 4 (A) Quantitative analysis of apoptotic U87R cells using Annexin V-FITC/PI double-staining assay and ow cytometry at 48 h after different treatments. (B)
Statistical analysis of apoptotic U87R cells. The dose of siPLK1 was 1 µg mL
1
. Data are shown as mean ± standard deviation (n = 3), ***P< 0.001.
Figure 5 (A) The expression of PLK1 protein in U87R cells receiving different treatments by Western blot analysis. (B) The expression of MGMT protein in T98G, LN-229,
U87 and U87R cells by Western blot analysis. (C) The relative expression of PLK1 protein in U87R cells after different treatment by Western blot analysis. (D) The relative
expression of MGMT protein in T98G, LN-229, U87 and U87R cells by Western blot analysis. (E) The relative expression of PLK1 mRNA in U87R cells after treatment by
qRT-PCR. The concentration of siPLK1 was 1 µg mL
1
. Data are shown as mean ± standard deviation (n = 3), *P< 0.05, **P< 0.01, ***P< 0.001.
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to the TMZ (Figure 5D). These results suggested that the
establishment of TMZ-resistant cell line U87R was successful.
In vitro Cell Viability Assay
The in vitro cell viability in U87R and LN-229 glioma
cells was evaluated using MTT assays. In the U87R cells
(Figure 6A), the cell viability of the TMZ-A2PEC
/siPLK1 group (23%) was obviously lower than that of
the free TMZ (50%) and TMZ-A2PEC/NCsiRNA groups
(48%). However, the cell viability of cells treated with
the free siPLK1 and A2PEC/siPLK1 were 89% and 84%,
respectively. The results in the LN-229 cells (Figure 6B)
were consistent with those in the U87R cells. These
results showed that combined treatment with TMZ and
siPLK1, mediated by A2-targeted NPs, yielded higher
cytotoxicity than TMZ therapy alone.
Antitumor Therapy Efcacy
The ability of NPs loaded with TMZ and siPLK1 to cross the
BBB and enter into glioma tissues is crucial for the anti-
glioma therapy. For this purpose, the brain glioma models
were constructed by intracranial injection of 8 × 10
5
U87-
Luci cells into male nude mice. In vivo bioluminescence
imaging demonstrated the existence of brain glioma
(Figure 7A). The nude mice were injected with PBS, free
DiR, and A2PEC/Dir through the tail vein, respectively.
After 4 h, in vivo uorescence imaging was performed
(Figure 7B and 7C). The strongest DiR uorescence was
observed in the gliomas of the mice injected with A2PEC/
DiR, indicating the accumulation of A2PEC/DiR in the
brain gliomas. Meanwhile, little DiR uorescence was
observed after the mice were injected with free DiR. These
results indicated that A2PEC could help TMZ and siPLK1
cross the BBB and enter into gliomas effectively.
The tissue biodistribution was examined after the
nude mice were injected with PBS, free DiR, and
A2PEC/DiR through the tail vein for 4 h, respectively
(Figure 7D and 7E). The results showed that both the
free DiR and A2PEC/DiR underwent a rapid and wide-
spread distribution in the liver and kidney tissues within
4 h. However, the uorescence intensity in the liver and
kidney in the mice treated with A2PEC/DiR was sig-
nicantly higher than that in mice treated with free DiR.
These results showed that vector A2PEC can exten-
sively prolong the retention time of serum siPLK1, and
this increased circulation time would provide TMZ-
A2PEC/siPLK1 with a better chance of accumulating
in brain gliomas.
To further verify the efcacy of NPs in vivo, we
constructed a glioma model using U87-Luci cells. The
mice were divided into six groups (n = 6), and received
PBS, free TMZ, free siPLK1, A2PEC/siPLK1, TMZ-
A2PEC/NCsiRNA, and TMZ-A2PEC/siPLK1 injected
via their tail vein, respectively (the dose of siPLK1
was 1 mg kg
1
). The U87-Luci tumor-bearing mice
were treated four times during the whole experiment
(Figure 8A). The bioluminescence intensity was used
to measure the glioma growth in the mice (Figure 8B).
Tumors in the control group (PBS) grew rapidly.
By day 30, the tumors in the control group, as reected
by the bioluminescence measurements (Figure 8C),
were 26-fold larger than those at day 10. Following
the control group, the bioluminescence measurements
of free siPLK1 and A2PEC/siPLK1 groups at day 30
Figure 6 MTT assay of U87R (A) and LN-229 (B) glioma cells. Data are shown as mean ± standard deviation (n = 3), ***P< 0.001.
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were 22-fold and 20-fold higher than those at day 10,
respectively. The tumor growth rate in the mice that
were treated with free TMZ, TMZ-A2PEC/NCsiRNA,
and TMZ-A2PEC/siPLK1 were 7.8-fold, 5.7-fold and
1.4-fold higher than at day 10, respectively. This result
indicated that TMZ-A2PEC/siPLK1 exhibited higher
anti-glioma activity than the other treatments, and indi-
cated that the combination of TMZ and siPLK1 could
effectively enhance TMZ sensitivity to treat glioma.
To further estimate antitumor efcacy, the U87-Luci
tumor-bearing mice were monitored for body weight change
andmediansurvivaltime(Figure 8D and 8E). The median
survival times in the PBS, free siPLK1, A2PEC/siPLK1, free
TMZ, TMZ-A2PEC/NCsiRNA, and TMZ-A2PEC/siPLK1
groups were 36, 36.5, 38.5, 41.5, 43, and 47.5 days, respec-
tively (Figure 8E). The survival time of U87-Luci tumor-
bearing mice treated with the TMZ-A2PEC/siPLK1 was
signicantly prolonged. These results indicated that TMZ-
A2PEC/siPLK1 could effectively inhibit the growth of
glioma. The dominance of TMZ-A2PEC/siPLK1 was also
reected by comparing the changes in body weight. The
body weight of the mice treated with TMZ-A2PEC/siPLK1
decreased slowly decreased, while all other groups lost
weight rapidly (Figure 8D).
The toxicity of the different formulations toward
normal tissues (heart, liver, spleen, lung, and kidney)
was investigated (Figure 9). Compared with the control
group, the H&E staining images of the major organs in
the treatment groups indicated that no noticeable tissue
damage or obvious changes in morphology. These
results suggested that the combination of TMZ and
siPLK1 delivered by the drug-loaded NP is effective in
treating glioma and did not induce systemic toxicity in
the tumor-bearing mice.
Figure 7 Distribution of Dir after intravenous injection of mice with PBS, free Dir and A2PEC/Dir in U87-Luci tumor-bearing mice. (A) Bioluminescence of luciferase
expressing tumor cells 10 min after injection with luciferin solution. (B) In vivo imaging uorescence signal of Dir. (C) Quantitative analysis of Dir in the mouse brains. Data
are presented as mean ± standard deviation (n = 4). (D) Tissue biodistribution of PBS, free Dir, and A2PEC/Dir in U87-Luci tumor-bearing mice after tail vein injection. (E)
Quantitative analysis of Dir in the mouse tissues.
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Discussion
Glioma is the most common type of primary CNS tumor,
accounting for about 80% of all malignant brain tumors.
3942
Although treatment using surgery, postoperative chemother-
apy, and radiotherapy have advanced signicantly, patients
prognosis is still poor, with an average survival rate of
approximately one year after diagnosis.
4347
TMZ is the
rst line chemotherapy for glioma. Although it is used widely
to treat patients with glioma, its effectiveness in treatment is
not very high because of TMZ resistant glioma.
48,49
PLK1 is
a serine/threonine protein kinase that is an early trigger for
G2/M conversion in the cell cycle.
50
Inhibition of PLK1
expression can arrest the cell cycle in the G2/M phase,
thereby increasing the effectiveness of chemotherapy
drugs.
27,28
Combining a PLK1 inhibitor and chemotherapy
drugs has already been reported, and can alleviate the drug-
resistance of tumor cells.
27
To sensitize glioma to TMZ, we
devised a vector that could co-deliver TMZ and siPLK1, and
both the in vivo and in vitro experiments showed that the
effectiveness of TMZ treatment of glioma improved
signicantly.
In the present study, the copolymer A2PEC was used
as the nanocarrier, with TMZ encapsulated in the core
through intermolecular hydrophobic interactions and
siPLK1 attached to the cationic layer via electrostatic
complexation. The average particle size and the zeta
potential of TMZ-A2PEC/siPLK1 were 82.22 nm and
18.80 mV, respectively. These results indicated that TMZ-
A2PEC/siPLK1 was suitable for brain targeted drug deliv-
ery. Besides, A2 was used to modify the nanocarrier to
facilitate the cellular uptake of the codelivery system. The
results showed that this nanocarrier provided remarkable
efcacy in co-delivering TMZ and siPLK1 into U87R
cells, and the A2 modied nanocomplexes had a much
better delivery efciency than the non-targeted ones. The
CLSM results showed that the NPs had escaped from the
Figure 8 In vivo antitumor therapy efcacy in the U87-Luci glioma mouse model. (A) U87-Luci tumor-bearing mice received four injection of PBS, free siPLK1, A2PEC/
siPLK1, free TMZ, TMZ-A2PEC/NCsiRNA, TMZ-A2PEC/siPLK1 at the dose of siPLK1 is 1 mg kg-1 on days 12, 14, 16, and 18. (B) Bioluminescent signal of U87-Luci tumor-
bearing mice of each groups after treatment. (C) Quantication of the tumor bioluminescence signal (n = 4). (D) Body weight change of the U87-Luci tumor-bearing mice (n
= 6). (E) KaplanMeier survival curve for the mice. Data are presented as mean ± standard deviation (n = 6), **P< 0.01.
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endosome after the U87R cells were treated with A2PEC/
FAM-siRNA for 4 h, which would result in enhanced gene
silencing by siPLK1.
Gene silencing ability is the most important factor
in siRNA therapies.
33
Our results of qRT-PCR and
Western blotting showed that TMZ-A2PEC/siPLK1
could silence PLK1 gene expression signicantly, lead-
ing to downregulated PLK1 protein levels in glioma
cells. Besides, the MGMT protein level in U87R cells,
whichisconsistentwithdrugresistancetoTMZ,was
signicantly higher than that in the U87 and LN-229
cells, suggesting the successful establishment of the
TMZ-resistant cell line U87R. The results were consis-
tent with those of Yang et al.
51
PLK1 is an early
trigger for G2/M conversion in the cell cycle, and
whether the cell cycle can arrest in the G2/M phase
by silencing the gene is a crucial factor.
24,25
In this
study, the results of the cell cycle arrest experiment
showed that both U87R and LN-229 cells developed
G2/M arrest, and the percentages of cells in G2/M that
had been treated with TMZ-A2PEC/siPLK1 were
40.33% and 43.49%, respectively. The MTT and apop-
tosis assays showed that the combined treatment with
TMZ and siPLK1 had a potent effect on cell apoptosis
by suppressing the expression of PLK1, which resulted
in an elevated in vitro anticancer effect.
To study the targeting effect to U87 cells, we con-
structed the U87-transplanted tumor model. Fluorescence
imaging was performed after injecting DiR into the tail
vein of the U87-Luci tumor-bearing mice. The results
demonstrated that A2PEC could cross the BBB, and
mainly accumulated in the liver and kidney. An in vivo
study showed that the tumor growth rate in the mice
treated with TMZ-A2PEC/siPLK1 was the lowest among
all the groups. The survival time of U87-Luci tumor-
bearing mice treated with the TMZ-A2PEC/siPLK1 was
prolonged signicantly and the body weight of these mice
showed the slowest decrease in all groups. The results
indicated that co-delivery of TMZ and siPLK1 using
A2PEC could effectively enhance TMZ sensitivity, and
signicantly inhibit glioma growth. Moreover, the H&E
staining images of major organs in the mice indicated that
Figure 9 H&E staining of heart, liver, spleen, lung, and kidney of the U87-Luci tumor-bearing mice treated with PBS, free siPLK1, A2PEC/siPLK1, free TMZ, TMZ-A2PEC
/NCsiRNA, TMZ-A2PEC/siPLK1 at the dose of siPLK1 is 1 mg kg
1
(scale bar = 200 µm).
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TMZ-A2PEC/siPLK1 caused no signicant damage to
other tissues and organs. Therefore, TMZ-A2PEC
/siPLK1 has the potential to treat glioma.
In conclusion, we successfully prepared a safe and
highly effective A2-modied nanocarrier, A2PEC, for the
codelivery of TMZ and siPLK1. Both in vitro and in vivo
studies demonstrated remarkably efcient co-delivery of
the two therapeutic agents into glioma cells. Consequently,
the PLK1 gene was effectively silenced, and the cell cycle
of glioma cells was arrested in the G2/M, which improved
the treatment effect of TMZ and led to increased tumor
cell apoptosis. The animal study proved that the combined
therapy of TMZ and siPLK1 mediated by A2-modied
nanocarriers, inhibited tumor growth markedly and drama-
tically prolonged the survival time of glioma tumor-
bearing mice. Thus, co-loading of TMZ and siPLK1 into
A2PEC NPs could be an effective targeted delivery strat-
egy for glioma treatment, reversing TMZ resistance and
improving therapeutic efcacy.
Acknowledgments
This work was supported by a grant from the National
Natural Science Foundation of China (No. 81772665).
This paper was also nanced from Natural Science
Foundation of Jiangsu Province (No. BK20150221),
Jiangsu Provincial Commission of Health and Family
Planning (No. Q201608). Research was supported by
Six Talents Peak Foundation of Jiangsu Province (No.
2018-WSW-071), Research and Development Fund
Project of Kangda College afliated to Nanjing
Medical University (No. KD2018KYJJYB048), Five-
two-one Project Research Fund of Lianyungang (No.
LYG52105-2018063).
Author Contributions
Conception and design: R. Yu, H. Liu, J. Lu, H. Shi.
Development of methodology: S. Sun, H. Xu. Acquisition
of data (provided animals, acquired and managed patients,
provided facilities, etc.): H. Shi, S. Sun, Z. Zhao, J. Jia.
Analysis and interpretation of data (eg, statistical analysis,
biostatistics, computational analysis): H. Shi, Z. Han,
Z. Zhao, J. Jia. Writing, review, and/or revision of the manu-
script: H. Shi, S. Sun, R. Yu, H. Liu. Administrative, tech-
nical, or material support (ie, reporting or organizing data,
constructing databases): J. Lu, D. Wu, R. Yu, H. Liu. All
authors contributed to data analysis, drafting or revising the
article, gave nal approval of the version to be published,
and agree to be accountable for all aspects of the work.
Disclosure
No conicts of interest were disclosed.
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... [435,436] Further surface modifications allow BCP assemblies to enhance the cell penetration and BBB permeability. [437,438] For instance, Kanazawa and co-workers loaded siRNA against TNF-into a composite of PEG-PCL-Tat, which was formed by conjugating the PEG-PCL micelles with a targeting peptide Tat. siRNA delivered by the established nanocarriers through intranasal administration lowered tTNF-production and neurological score of a rat model of cerebral stroke. ...
... [439] Another study carried out by Shi et al. showed that angiopep-2 (A2)-modified PEG-PEI-PCL micelles can deliver siRNA and drugs to brain tumor both in vitro and in vivo. [438] Additionally, the polymersomes have been also used to the codelivery of nucleic acids and other smaller NPs to achieve multiple functions. For example, Lu et al. developed a polymersome to codeliver superparamagnetic iron oxide NPs and siRNA into the NSC, which were subsequently transplanted into the striatum ipsilateral of the ischemic rat brain. ...
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Central Nervous System (CNS) diseases, such as Alzheimer's diseases (AD), Parkinson's Diseases (PD), brain tumors, Huntington's disease (HD), and stroke, still remain difficult to treat by the conventional molecular drugs. In recent years, various gene therapies have come into the spotlight as versatile therapeutics providing the potential to prevent and treat these diseases. Despite the significant progress that has undoubtedly been achieved in terms of the design and modification of genetic modulators with desired potency and minimized unwanted immune responses, the efficient and safe in vivo delivery of gene therapies still poses major translational challenges. Various non‐viral nanomedicines have been recently explored to circumvent this limitation. In this review, an overview of gene therapies for CNS diseases is provided and describes recent advances in the development of nanomedicines, including their unique characteristics, chemical modifications, bioconjugations, and the specific applications that those nanomedicines are harnessed to deliver gene therapies.
... The standard treatment for glioma is surgical resection combined with radiotherapy and chemotherapy. However, most gliomas recur or progress, and the long-term effect of treatments is limited due to their highly invasive and infiltrative features (4,6). ...
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Background Glioma is a highly aggressive brain cancer with a poor prognosis. Necroptosis is a form of programmed cell death occurring during tumor development and in immune microenvironments. The prognostic value of necroptosis in glioma is unclear. This study aimed to develop a prognostic glioma model based on necroptosis.MethodsA necroptosis-related risk model was constructed by Cox regression analysis based on The Cancer Genome Atlas (TCGA) training set, validated in two Chinese Glioma Genome Atlas (CGGA) validation sets. We explored the differences in immune infiltration and immune checkpoint genes between low and high risk groups and constructed a nomogram. Moreover, we compiled a third validation cohort including 43 glioma patients. The expression of necroptosis-related genes was verified in matched tissues using immunochemical staining in the third cohort, and we analyzed their relationship to clinicopathological features.ResultsThree necroptosis-related differentially expressed genes (EZH2, LEF1, and CASP1) were selected to construct the prognostic model. Glioma patients with a high risk score in the TCGA and CGGA cohorts had significantly shorter overall survival. The necroptosis-related risk model and nomogram exhibited good predictive performance in the TCGA training set and the CGGA validation sets. Furthermore, patients in the high risk group had higher immune infiltration status and higher expression of immune checkpoint genes, which was positively correlated with poorer outcomes. In the third validation cohort, the expression levels of the three proteins encoded by EZH2, LEF1, and CASP1 in glioma tissues were significantly higher than those from paracancerous tissues. They were also closely associated with disease severity and prognosis.Conclusions Our necroptosis-related risk model can be used to predict the prognosis of glioma patients and improve prognostic accuracy, which may provide potential therapeutic targets and a theoretical basis for treatment.
... Both in vitro and in vivo studies showed that the simultaneous release of anti-cancer drugs and therapeutic genes to cervical and gastric cancer cells led to the inhibition of tumor growth and proliferation [15,39,40]. Approaches to address glioblastoma therapy based on drug/gene co-delivery, namely on the use of TMZ combined with pDNA or siRNA, have already been demonstrated as providing enhanced therapeutic effects [41][42][43]. Despite some studies available in the literature, this chemo/gene therapy combination has been poorly explored but it should be deeply investigated for improved outcomes in glioblastoma treatment. ...
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Despite the great progress over the past few decades in both the diagnosis and treatment of a great variety of human cancers, glioblastoma remains the most lethal brain tumor. In recent years, cancer gene therapy focused on non-viral vectors which emerged as a promising approach to glioblastoma treatment. Transferrin (Tf) easily penetrates brain cells of the blood–brain barrier, and its receptor is highly expressed in this barrier and glioblastoma cells. Therefore, the development of delivery systems containing Tf appears as a reliable strategy to improve their brain cells targeting ability and cellular uptake. In this work, a cell-penetrating peptide (WRAP5), bearing a Tf-targeting sequence, has been exploited to condense tumor suppressor p53-encoding plasmid DNA (pDNA) for the development of nanocomplexes. To increase the functionality of developed nanocomplexes, the drug Temozolomide (TMZ) was also incorporated into the formulations. The physicochemical properties of peptide/pDNA complexes were revealed to be dependent on the nitrogen to phosphate groups ratio and can be optimized to promote efficient cellular internalization. A confocal microscopy study showed the capacity of developed complexes for efficient glioblastoma cell transfection and consequent pDNA delivery into the nucleus, where efficient gene expression took place, followed by p53 protein production. Of promise, these peptide/pDNA complexes induced a significant decrease in the viability of glioblastoma cells. The set of data reported significantly support further in vitro research to evaluate the therapeutic potential of developed complexes against glioblastoma.
... To this end, PLK1 is reported to increase the temozolomide sensitivity in glioma stem cells [91]. Recently, the combination of temozolomide and PLK1 inhibitor has shown synergistic cytotoxicity in glioma cells in vivo [92]. USP9X (ubiquitin-specific peptidase 9 X-linked) is a deubiquitinase which regulates the protein levels of its substrates through proteasomal degradation. ...
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Chromatin remodeling is an essential cellular process for organizing chromatin structure into either open or close configuration at specific chromatin locations by orchestrating and modifying histone complexes. This task is responsible for fundamental cell physiology including transcription, DNA replication, methylation, and damage repair. Aberrations in this activity have emerged as epigenomic mechanisms in cancer development that increase tumor clonal fitness and adaptability amidst various selection pressures. Inactivating mutations in AT-rich interaction domain 1A ( ARID1A ), a gene encoding a large nuclear protein member belonging to the SWI/SNF chromatin remodeling complex, result in its loss of expression. ARID1A is the most commonly mutated chromatin remodeler gene, exhibiting the highest mutation frequency in endometrium-related uterine and ovarian carcinomas. As a tumor suppressor gene, ARID1A is essential for regulating cell cycle, facilitating DNA damage repair, and controlling expression of genes that are essential for maintaining cellular differentiation and homeostasis in non-transformed cells. Thus, ARID1A deficiency due to somatic mutations propels tumor progression and dissemination. The recent success of PARP inhibitors in treating homologous recombination DNA repair-deficient tumors has engendered keen interest in developing synthetic lethality-based therapeutic strategies for ARID1A -mutated neoplasms. In this review, we summarize recent advances in understanding the biology of ARID1A in cancer development, with special emphasis on its roles in DNA damage repair. We also discuss strategies to harness synthetic lethal mechanisms for future therapeutics against ARID1A -mutated cancers.
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Glioblastoma multiforme (GBM) is one of the most lethal cancer due to poor diagnosis and rapid resistance developed towards the drug. Genes associated to cancer-related overexpression of proteins, enzymes, and receptors can be suppressed using an RNA silencing technique. This assists in obtaining tumour targetability, resulting in less harm caused to the surrounding healthy cells. RNA interference (RNAi) has scientific basis for providing potential therapeutic applications in improving GBM treatment. However, the therapeutic application of RNAi is challenging due to its poor permeability across blood–brain barrier (BBB). Nanobiotechnology has evolved the use of nanocarriers such as liposomes, polymeric nanoparticles, gold nanoparticles, dendrimers, quantum dots and other nanostructures in encasing the RNAi entities like siRNA and miRNA. The review highlights the role of these carriers in encasing siRNA and miRNA and promising therapy in delivering them to the glioma cells.
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Central nervous system (CNS) diseases are among the most difficult to treat, mainly because the vast majority of the drugs fail to cross the blood-brain barrier (BBB) or to reach the brain at concentrations adequate to exert a pharmacological activity. The obstacle posed by the BBB has led to the in-depth study of strategies allowing the brain delivery of CNS-active drugs. Among the most promising strategies is the use of peptides addressed to the BBB. Peptides are versatile molecules that can be used to decorate nanoparticles or can be conjugated to drugs, with either a stable link or as pro-drugs. They have been used to deliver to the brain both small molecules and proteins, with applications in diverse therapeutic areas such as brain cancers, neurodegenerative diseases and imaging. Peptides can be generally classified as receptor-targeted, recognizing membrane proteins expressed by the BBB microvessels (e.g., Angiopep2, CDX, and iRGD), "cell-penetrating peptides" (CPPs; e.g. TAT47-57, SynB1/3, and Penetratin), undergoing transcytosis through unspecific mechanisms, or those exploiting a mixed approach. The advantages of peptides have been extensively pointed out, but so far few studies have focused on the potential negative aspects. Indeed, despite having a generally good safety profile, some peptide conjugates may display toxicological characteristics distinct from those of the peptide itself, causing for instance antigenicity, cardiovascular alterations or hemolysis. Other shortcomings are the often brief lifetime in vivo, caused by the presence of peptidases, the vulnerability to endosomal/lysosomal degradation, and the frequently still insufficient attainable increase of brain drug levels, which remain below the therapeutically useful concentrations. The aim of this review is to analyze not only the successful and promising aspects of the use of peptides in brain targeting but also the problems posed by this strategy for drug delivery.
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Selective targeting of elevated copper (Cu) in cancer cells by chelators to induce tumor-toxic reactive oxygen species (ROS) may be a promising approach in the treatment of glioblastoma multiforme (GBM). Previously, the Cu chelator di-2-pyridylketone-4,4-dimethyl-3-thiosemicarbazone (Dp44mT) attracted much interest due to its potent anti-tumor activity mediated by the formation of a highly redox-active Cu-Dp44mT complex. However, its translational potential was limited by the development of toxicity in murine models of cancer reflecting poor selectivity. Here, we overcame the limitations of Dp44mT by incorporating it in new biomimetic nanoparticles (NPs) optimized for GBM therapy. Biomimetic design elements enhancing selectivity included angiopeptide-2 functionalized red blood cell membrane (Ang-M) camouflaging of the NPs carrier. Co-loading Dp44mT with regadenoson (Reg), that transiently opens the blood-brain-barrier (BBB), yielded biomimetic Ang-MNPs@(Dp44mT/Reg) NPs that actively targeted and traversed the BBB delivering Dp44mT specifically to GBM cells. To further improve selectivity, we innovatively pre-loaded GBM tumors with Cu. Oral dosing of U87MG-Luc tumor bearing mice with diacetyl-bis(4-methylthiosemicarbazonato)-copperII (Cu(II)-ATSM), significantly enhanced Cu-level in GBM tumor. Subsequent treatment of mice bearing Cu-enriched orthotopic U87MG-Luc GBM with Ang-MNPs@(Dp44mT/Reg) substantially prevented orthotopic GBM growth and led to maximal increases in median survival time. These results highlighted the importance of both angiopeptide-2 functionalization and tumor Cu-loading required for greater selective cytotoxicity. Targeting Ang-MNPs@(Dp44mT/Reg) NPs also down-regulated antiapoptotic Bcl-2, but up-regulated pro-apoptotic Bax and Cleaved-caspase-3, demonstrating the involvement of the apoptotic pathway in GBM suppression. Notably, Ang-MNPs@(Dp44mT/Reg) showed negligible systemic drug toxicity in mice, further indicating therapeutic potential that could be adapted for other central nervous system disorders.
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Glioblastoma (GBM) is an aggressive, fatal and malignant primary brain tumor. Despite the current standard treatment for glioblastoma patients including neurosurgical resection, followed by concomitant radiation and chemotherapy, the median survival rate is about 15 months. An unresolved challenge for current therapies is related to getting drugs through the blood-brain barrier (BBB), which hinders many chemotherapeutic agents from reaching tumors cells. Although a large amount of research has been done to circumvent the BBB and deliver drugs to the brain, with nanoparticles (NPs) taking the lead, the challenge is still high. In this regard, the BBB and how to transfer drug pathways through the BBB, especially using NPs, are introduced here. Afterwards, the latest advances in drug delivery, co-drug delivery, and combination modality are described specifically for GBM treatment using natural and synthetic polymeric NPs and adjuvant therapies including hyperthermia, photodynamic therapy and also ketogenic regimens. In addition, receptor-mediated endocytosis agents that exist in endothelial capillary cells of the brain are explained. Lastly, future directions to finally deliver drugs through the BBB for GBM treatment are emphasized. It is the hope that this review can provide a number of practical pathways for the future development of BBB permeable nanochemotherapeutics against GBM.
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Malignant gliomas are the most aggressive forms of brain tumors; whose metastasis and recurrence contribute to high rates of morbidity and mortality. Glioma stem cell-like cells are a subpopulation of tumor-initiating cells responsible for glioma tumorigenesis, metastasis, recurrence and resistance to therapy. Epidermal growth factor receptor (EGFR) has been reported to be dysregulated in most cancers, including gliomas and its functions are closely linked to initiating tumor metastasis and a very poor prognosis. In search for compounds that may reduce the tumorigenic potential of gliomas/glioblastomas honokiol attracted our attention. Honokiol, purified from the bark of traditional Chinese herbal medicine Magnolia species, is beneficial in vitro and in animal models via a variety of pharmacological effects, including anti-inflammatory, anti-angiogenetic, anti-arrhythmic and antioxidant activities, as well as anti-proliferative and proapoptotic effects in a wide range of human cancer cells. However, its effects on glioma cells are unknown. Here, we used different concentrations of honokiol in treating U251 and U-87 MG human glioma/glioblastoma cells in cell culture. Results showed that honokiol inhibited glioma cell viability and colony formation and promoted apoptosis. It also inhibited glioma cell migration/proliferation and invasion. In addition, honokiol promoted apoptosis and reduced Bcl-2 expression, accompanied by increase in Bax expression. Honokiol reduced expression of EGFR, CD133 and Nestin. Moreover, honokiol inhibited the activation of both AKT and ERK signaling pathways, increased active caspase-3 level and reduced phosphorylation of STAT3. U-87 MG xenografts in nude mice and in immunotolerant zebrafish yolk sac showed that honokiol inhibits tumor growth and metastasis. Altogether, results indicate that honokiol reduces tumorigenic potentials, suggesting hopes for honokiol to be useful in the clinical management of glioma/glioblastoma.
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Background Glioma is the most common highly aggressive, primary adult brain tumour. Clinical data show that therapeutic approaches cannot reach the expectations in patients, thus gliomas are mainly incurable diseases. Tumour cells can adapt rapidly to alterations during therapeutic treatments related to their metabolic rewiring and profound heterogeneity in tissue environment. Renewed interests aim to develop effective treatments targeting angiogenesis, kinase activity and/or cellular metabolism. mTOR (mammalian target of rapamycin), whose hyper-activation is characteristic for many tumours, promotes metabolic alterations, macromolecule biosynthesis, cellular growth and survival. Unfortunately, mTOR inhibitors with their lower toxicity have not resulted in appreciable survival benefit. Analysing mTOR inhibitor sensitivity, other metabolism targeting treatments and their combinations could help to find potential agents and biomarkers for therapeutic development in glioma patients. Methods In vitro proliferation assays, protein expression and metabolite concentration analyses were used to study the effects of mTOR inhibitors, other metabolic treatments and their combinations in glioma cell lines. Furthermore, mTOR activity and cellular metabolism related protein expression patterns were also investigated by immunohistochemistry in human biopsies. Temozolomide and/or rapamycin treatments altered the expressions of enzymes related to lipid synthesis, glycolysis and mitochondrial functions as consequences of metabolic adaptation; therefore, other anti-metabolic drugs (chloroquine, etomoxir, doxycycline) were combined in vitro. Results Our results suggest that co-targeting metabolic pathways had tumour cell dependent additive/synergistic effects related to mTOR and metabolic protein expression patterns cell line dependently. Drug combinations, especially rapamycin + doxycycline may have promising anti-tumour effect in gliomas. Additionally, our immunohistochemistry results suggest that metabolic and mTOR activity alterations are not related to the recent glioma classification, and these protein expression profiles show individual differences in patients’ materials. Conclusions Based on these, combinations of different new/old drugs targeting cellular metabolism could be promising to inhibit high adaptation capacity of tumour cells depending on their metabolic shifts. Relating to this, such a development of current therapy needs to find special biomarkers to characterise metabolic heterogeneity of gliomas.
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