Evaluation of novel paclitaxel-loaded NO-donating polymeric micelles for an improved therapy for gastroenteric tumor

Article abstract of DOI:10.1039/d1nj00979f

This study reports the design and synthesis of NO-donating polymer to generate biodegradable polymeric micelle containing paclitaxel (NO/PTX) as a nanomedicine delivery system aimed to enhance the solubility and anti-cancer activity of paclitaxel (PTX). NO/PTX showed greater NO-releasing performance than nitroglycerin, displaying excellent tolerance in KM mice, and exhibited a two-fold stronger antiproliferative activity than PTXin vitroagainst HCT116, SW480, and SGC-7901 cell lines.In vivotumor growth inhibition assay results indicated that NO/PTX displayed lightly stronger activities against tumor growth than PTX at a dose of 10 mg kg^?1, while the anti-tumor effect of NO/PTX was significantly improved than that of PTX and Genexol-PM groups at a dose of 15 mg kg^?1(the inhibition rate: 67%vs.53% and 41%). In addition, NO/PTX showed an improved area under the plasma concentration-time curve and drug deposition in tumors in comparison to PTX. Wound healing assay and western blot analysis of EMT-related markers suggested that NO/PTX could inhibit the potential of HCT116 migration. Western blot analysis also demonstrated that NO/PTX dampened efflux activity of P-gp and up-regulated apoptosis-related proteins. Overall, these promising results suggested that the synergism between PTX and NO-donating micelles could contribute to the potent anti-cancer activity of NO/PTX.

Full text of DOI:10.1039/d1nj00979f

Showcasing research from the Zunhua Yang lab at the
Jiangxi University of Chinese Medicine, China.
As featured in:
Volume 45
Number 31
21 August 2021
Pages 13651–14240
Evaluation of novel paclitaxel-loaded NO-donating
polymeric micelles for an improved therapy for
gastroenteric tumor
NJC
New Journal of Chemistry
A
journal for new directions in chemistry
rsc.li/njc
A NO-releasing polymer (mPEG-PLA-NO) is developed
as micellar nanoparticle delivery system for the carrier of
paclitaxel. The amphiphilic copolymer will self-assemble to
form a paclitaxel-loaded micelle to release paclitaxel and
NO in tumor cells and to exert improved water-solubility,
enhanced antitumor effect, reduced therapeutical toxicity
and anti-metastasis potential.
ISSN 1144-0546
PAPER
Priyanka Srivastava, Ute Resch-Genger et al.
Synthesis and spectroscopic characterization of
fluorescent phenanthrene-rhodamine dyad for ratiometric
measurements of acid pH values
a
See Zunhua Yang et al.,
New J. Chem., 2021, 45, 13763.
rsc.li/njc
Registered charity number: 207890

NJC
View Article Online
View Journal | View Issue
PAPER
Evaluation of novel paclitaxel-loaded
NO-donating polymeric micelles for an improved
therapy for gastroenteric tumor
Cite this: New J. Chem., 2021,
45, 13763
Huilan Li,†a Yuanying Fang, †b Xiang Li,b Liangxing Tu,a Guoliang Xu,a Yi Jin,b
a
Ronghua Liua and Zunhua Yang
*
This study reports the design and synthesis of NO-donating polymer to generate biodegradable
polymeric micelle containing paclitaxel (NO/PTX) as a nanomedicine delivery system aimed to enhance the
solubility and anti-cancer activity of paclitaxel (PTX). NO/PTX showed greater NO-releasing performance
than nitroglycerin, displaying excellent tolerance in KM mice, and exhibited
a two-fold stronger
antiproliferative activity than PTX in vitro against HCT116, SW480, and SGC-7901 cell lines. In vivo tumor
growth inhibition assay results indicated that NO/PTX displayed lightly stronger activities against tumor
growth than PTX at a dose of 10 mg kgÀ1, while the anti-tumor effect of NO/PTX was significantly
improved than that of PTX and Genexols-PM groups at a dose of 15 mg kgÀ1 (the inhibition rate: 67% vs.
53% and 41%). In addition, NO/PTX showed an improved area under the plasma concentration-time curve
and drug deposition in tumors in comparison to PTX. Wound healing assay and western blot analysis of
EMT-related markers suggested that NO/PTX could inhibit the potential of HCT116 migration. Western
blot analysis also demonstrated that NO/PTX dampened efflux activity of P-gp and up-regulated
apoptosis-related proteins. Overall, these promising results suggested that the synergism between PTX
and NO-donating micelles could contribute to the potent anti-cancer activity of NO/PTX.
Received 26th February 2021,
Accepted 25th June 2021
DOI: 10.1039/d1nj00979f
rsc.li/njc
using monomethoxy poly(ethylene glycol)-polylactide (mPEG-PLA)
1. Introduction
as a carrier material,19 The in vivo antitumor efficacy of Genexols-
Paclitaxel (PTX) is a well-known anti-cancer drug used for treatment PM against SKOV-3 human ovarian cancer and MX-1 human
including breast cancer, ovarian cancer, and leukemia.1–3 However, breast cancer is significantly greater than that of Taxols.
due to weak solubility and adverse effects, PTX has limitations and
Numerous studies have provided evidence that the high
its clinical applications have to be further investigated;4–6 aimed to concentration of nitric oxide (NO) would generate a significant
increase the solubility and bioavailability of PTX, cremophor EL anti-tumor effect.20–23 NO concentration of more than 400 nM
was added to prepare formulations as an aqueous vehicle. could activate the p53 pathway, leading to cell cycle arrest and
Unfortunately, cremophor EL is known to induce hypersensitivity cell growth inhibition. When the concentration of NO surpassed
and nephrotoxicity.6,7 Thus, recently new pharmaceutical pre- 1 mM, the occurrence of nitrosation, nitration and alkylation
paration techniques such as liposomes,8,9 microspheres,10,11 would enhance the apoptosis of tumor cells by the repression of
nanocapsules,12,13 polymeric micelles14,15 and other carriers were specific DNA repair systems, causing disorders of energy meta-
developed as nanomedicine delivery systems to improve the water bolism and disruption of mitochondrial physiology.24,25 Further-
solubility, bioavailability and lower toxicity of taxanes. Especially, more, NO could improve the sensitivity of chemotherapy.26,27 NO
polymeric micelles have attracted more attention because of their donors would convert the anti-apoptotic NF-kB/Snail/YY1/RKIP/
unique structure and properties.16–18 Genexols-PM is the first PTEN resistant loop into a sensitive pro-apoptotic loop, inhibiting
paclitaxel polymeric micelle formulation approved by South Korea, tumor cell viability and proliferation, and sensitize cells to
apoptosis.28,29
NO donors could be divided structurally into several categories,
a College of Pharmacy, Jiangxi University of Traditional Chinese Medicine,
including organic nitrites and nitrates, metal nitrosyls, furoxans,
S-nitrosothiols, and diazeniumdiolates.30 Organic nitrate-based
Nanchang 330004, China. E-mail: joshyyy@126.com
b National Engineering Research Center for Manufacturing Technology of TCM Solid
NO donors were able to show anti-tumor activities by increasing
Preparation, Jiangxi University of Traditional Chinese Medicine, Nanchang
tumor cell oxygenation capacity and tumor perfusion to inhibit
tumor angiogenesis.29 Metal nitrosyl complexes could inhibit the
330006, China
† H. L. and Y. F. contributed equally to this work.
This journal is © The Royal Society of Chemistry and the Centre National de la Recherche Scientifique 2021
New J. Chem., 2021, 45, 13763–13774 | 13763

View Article Online
Paper
NJC
clone formation and cell invasion of tumor cells. Furoxan was a 2. Results and discussion
type of thiol-based heterocyclic NO donor. Thiol first attacked
2.1 Synthesis and characterization of NO donating polymer
position 3 or 4 of the furoxan ring, and then the ring was
dearomatized and opened to release NO, which could reverse
multiple drug resistance of tumor cells to make them more
sensitive to chemotherapy. Diazeniumdiolates had a long half-life
(20 hours at 37 1C) and could be used as long-acting NO donors.
Their mechanism of action was to interact with glutathione-S-
transferase in the human body and be catalyzed to produce NO,
and then come into an antitumor effect.31,32 NO donors could be
carried by polymeric nanoparticles to increase the circulation
stability and load amounts of NO in the solid tumor by the
enhanced permeability and retention effect.33–35
Therefore, in order to improve the anti-cancer efficacy, we
designed the NO donating micelle as a drug delivery system. We
supposed that the synergistic effect of PTX and NO might contribute
to the anti-cancer activity markedly, and nanomedicine-micelle
carrier could decrease the toxicity of PTX as well. The amphiphilic
copolymer is self-assembled to form a NO-donating paclitaxel-
loaded micelle, which would release PTX and NO in tumor cells
and exert an enhanced antitumor effect by inducing the apoptosis
of tumor cells (Fig. 1).
In this work, we developed a monomethoxy poly(ethylene
glycol)-polylactide (mPEG-PLA) micelles which were hybridized
with furoxan-based NO donor moiety and loaded with PTX to
generate a biodegradable polymeric micellar system (mPEG-
PLA-NO-PTX, abbreviated as NO/PTX). We then evaluated the
NO-releasing activity, acute toxicity, and antitumor efficacy
in vitro and in vivo, pharmacokinetics and tissue distribution,
wound healing activity, and effects on the expression of related
proteins, comparing with the current clinical formulation
of PTX.
To investigate the synergistic effect of NO for cancer therapy, we
developed a NO donating polymer as a nanoparticle carrier
of antitumor drugs. A monomethoxy poly(ethylene glycol)-
polylactide (mPEG-PLA, MW E 4 kDa) was firstly synthesized
by coupling mPEG2000 (MW E 2.2 kDa) with DL-lactide in the
presence of tin(II) octoate. The synthetic precursor furoxan was
prepared through 5 steps starting from thiophenol according to
the previous procedure.36 The NO donor furoxan was treated
with pivaloyl chloride to form a mixed anhydride, which was
coupled with mPEG-PLA to afford the ester mPEG-PLA-NO
(Scheme 1). The polymeric product was purified by fast filtration
from cold ethanol and dried in a vacuum.
1H NMR spectra of the polymer mPEG-PLA-NO were recorded
on a 600 MHz spectrometer using deuterated chloroform
(CDCl3) as a solvent and tetramethylsilane (TMS) as the internal
standard (Fig. 2). We could estimate the unit numbers of
monomer (n1 + 1 E 49, n2 + 1 E 27) according to the integrals
of featured peaks (d 3.65 (m, (4n1 + 2)H, –O–CH2CH2–O–), 5.14
À À À À
(m, (n2
+ 1)H, –OCOCH(CH3)–O–), 1.58(m, 3(n2 + 1)H,
À À
–OCOCH(CH3)–O–)). The molecular weight distributions (poly-
dispersity index, PDI = Mw/Mn) of mPEG-PLA-NO were deter-
À À
mined to be 1.13 by gel permeation chromatography (GPC).
2.2 Preparation of PTX-loaded micelle
The polymer mPEG-PLA-NO was dissolved in ethanol with the
help of surfactant HS15. PTX was also dissolved in the solution
after stirring. Mannitol solution was added to form the aqueous
micelle. The clear solution was lyophilized to afford the micelle
of PTX (NO/PTX).
The average diameter of NO/PTX micelles, determined by
dynamic light scattering (DLS), was less than 50 nm (Fig. 3A).
Transmission electron microscopy (TEM, Fig. 3B) investigation
showed that the micelles were spherical and had sizes consistent
with that of DLS measurements. The entrapment efficiency (EE%)
of PTX was determined to be 97.7 Æ 0.5%.35
2.3 NO-releasing test
The levels of NO release abilities from PTX and NO/PTX were
evaluated by the Griess method. Nitroglycerin and saline were
used as positive and negative controls. As shown in Fig. 3C,
while nitroglycerin released NO with a continuously growing
trend within 12 hours, NO/PTX displayed a greater release
performance than nitroglycerin with a peak at 6 hours. Only
PTX showed almost no release of nitric oxide.
2.4 Acute toxicity
The acute toxicity for mPEG-PLA, mPEG-PLA-NO, PTX and
NO/PTX were investigated in healthy male SPF KM mice. Four
groups of KM mice were injected with solutions via the caudal
vein at the dose of 1500, 2000 and 3200 mg kgÀ1 per day (mPEG-
PLA or mPEG-PLA-NO) or 20, 30, 36.3, 38.84, 40.55, 42.3 and
45 mg kgÀ1 per day (PTX) or 60, 80, 120, 141.72 and 160 mg kgÀ1
per day (NO/PTX) for seven days, respectively. Saline was used
Fig. 1 Proposed mechanism of action of NO/PTX micelles.
13764
|
New J. Chem., 2021, 45, 13763–13774
This journal is © The Royal Society of Chemistry and the Centre National de la Recherche Scientifique 2021

View Article Online
NJC
Paper
Scheme 1 Synthesis of the polymer mPEG-PLA-NO. Reagents and conditions: (a) ClCH2COOH, NaOH, reflux, 2 h; (b) 30% H2O2, CH3COOH, R.T., 2.5 h;
(c) fuming HNO3, 90 1C, 1.5 h; (d) 1,3-propanediol, 25% NaOH aq., THF, R.T., 3 h; (e) succinic anhydride, DMAP, DMF, 85 1C, 6 h; (f) tin(II) octoate, 130 1C,
18 h; (g) pivaloyl chloride, pyridine, DMAP, 0 1C to R.T., 36 h.
Fig. 2 1H NMR spectra of mPEG-PLA-NO in CDCl3.
2.5 In vitro cytotoxicity
as a control. The median lethal dose (LD50) and the maximum
non-lethal dose (MNLD) were determined by Graph Pad 6. As An MTT assay was used to evaluate the cytotoxicity of PTX and
summarized in Table 1, MNLD of mPEG-PLA and mPEG-PLA- NO/PTX against human colon cancer cell lines (HCT116, SW480
NO were 2000 mg kgÀ1 and 1500 mg kgÀ1, respectively, which and HT29), human gastric cancer cell line (SGC-7901) and
indicated that both mPEG-PLA and mPEG-PLA-NO were non- human breast cancer cell line (MCF-7). The concentrations
toxic amphiphilic copolymers. LD50 of PTX and NO/PTX in KM of PTX and NO/PTX were diluted 10-fold ranging from 0.0001
mice were determined to be 39.9 mg kgÀ1 and 137.1 mg kgÀ1
respectively. The acute toxicity of NO/PTX was reduced to 29% were calculated using the software SPSS 13.0. As illustrated in
of PTX.
Fig. 4A, NO/PTX exhibited two-fold more potent antiproliferative
,
to 10 mg mLÀ1. The 50% inhibitory concentration (IC50) values
This journal is © The Royal Society of Chemistry and the Centre National de la Recherche Scientifique 2021
New J. Chem., 2021, 45, 13763–13774 | 13765

View Article Online
Paper
NJC
Fig. 3 The size distribution of NO/PTX micelles by DLS (A) and TEM micrograph of NO/PTX micelles (B). NO releasing ability of NO/PTX micelles (n = 3)
(C). * p o 0.05 and ** p o 0.01 vs. saline group. #p o 0.05 and ##p o 0.01 vs. PTX group.
Table 1 Acute toxicities of mPEG-PLA, mPEG-PLA-NO, PTX and NO/PTX
in mice
2.7 In vivo tumor growth inhibition
To investigate the in vivo tumor growth inhibition of our PTX-
loaded NO-releasing polymeric micelle (NO/PTX), we compared
its antitumor effect with commercial PTX injection and Genexols-
PM (PTX-containing mPEG-PLA polymeric micellar system).
Firstly, we carried out the tumor growth inhibition assay in vivo
using the H22 hepatocellular carcinoma model in KM mice, for
H22 mice model could be conveniently available to demonstrate
the efficacy and toxicity of drug clearly. After the tumors were well-
established, PTX, Genexols-PM and NO/PTX were injected via the
caudal vein at doses of 10 and 15 mg kgÀ1 respectively on days 0,
3 and 6 with saline as the control. On day 9, mice were sacrificed
and the tumor was harvested and weighted. We also tested the
antitumor activity of the polymeric carriers mPEG-PLA and
mPEG-PLA-NO at the dose of 300 mg kgÀ1 in the same way.
The results are shown in Fig. 5. The polymer mPEG-PLA did
Compound
MNLD (mg kgÀ1
)
LD50 (mg kgÀ1
)
mPEG-PLA
mPEG-PLA-NO
PTX
2000
1500
36.3
80
—
—
39.9
137.1
NO/PTX
activities than PTX against HCT116, SW480 and SGC-7901, and
1.5 fold stronger activity toward MCF-7, but was less potent
against HT29. These preliminary results indicated that NO/PTX
displayed a more potent inhibitory effect with gastroenteric
cancer than PTX.
2.6 In vitro wound healing assay
Since a potent antiproliferative effect was observed against the
HCT116 cell line, HCT116 was selected for further studies. The not show any antitumor response. A slight antitumor effect was
inhibition of HTC116 migration by PTX and NO/PTX was assessed observed in the mPEG-PLA-NO group (Fig. 5B). Significant
using a wound-healing assay. The consistent width scratched antitumor activities were observed in PTX, Genexols-PM and
monolayer HCT116 cell line was treated with PTX or NO/PTX at NO/PTX groups. As illustrated in Table 2, PTX, Genexols-PM
concentrations of 0.01 and 0.1 mg mLÀ1. Images in different groups and NO/PTX groups showed comparable anti-tumor activities
were captured at 0, 12 and 24 h using a Leica DMI3008 inverted (inhibition rates were 39%, 36% and 41%, respectively) at a
microscope. Image J software was used to calculate the wound low dose (10 mg kgÀ1). Furthermore, when the dose was up to
healing area. Consequently, decreased cell motility was observed 15 mg kgÀ1, the anti-tumor effect of NO/PTX was significantly
in the HTC116 cell line treated with PTX or NO/PTX (Fig. 4B). The stronger than that of the PTX and Genexols-PM groups (the
migration potential of HTC116 cells was significantly inhibited by inhibition rates of PTX, Genexols-PM and NO/PTX group were
0.1 mg mLÀ1 of NO/PTX at 12 and 24 h in comparison with PTX.
53%, 41% and 67%, respectively, Fig. 5A), which proved that
13766
|
New J. Chem., 2021, 45, 13763–13774
This journal is © The Royal Society of Chemistry and the Centre National de la Recherche Scientifique 2021

View Article Online
NJC
Paper
Fig. 4 Cytotoxicity of PTX and NO/PTX against HCT116, SW480, HT29, SGC-7901 and MCF-7 cell lines (A). Inhibition of HTC116 cell motility by PTX or
NO/PTX (B).
the synergistic effect of NO and paclitaxel enhances the anti- determined by HPLC and the concentrations of PTX in the
tumor activity. When treated with the same dose of PTX or heart, liver, spleen, lungs and kidneys were determined using
NO/PTX, the mice lost their similar weight, which indicated HPLC-MS/MS with docetaxel as internal standard. The pharmaco-
that NO/PTX did not lead to extra toxicity in contrast to PTX.
kinetic parameters were processed by non-compartmental analysis
using the DAS 2.0 software package (from the Chinese Pharmaco-
logical Society).
2.8 Pharmacokinetics and tissue distribution
The pharmacokinetics and tissue distribution were studied in a
The pharmacokinetic parameters achieved are presented in
male KM mouse xenograft model established with H22 cells to Table 3. The levels of PTX in plasma at each time point in the
delineate the disposition of the NO/PTX in vivo. According to NO/PTX (50 mg kgÀ1) group were higher than PTX (20 mg kgÀ1
)
the positive reference Genexols-PM,19 we also use the same group (Fig. 6A) within the first four hours because the administered
dose to study the pharmacokinetics and tissue distribution. dose of NO/PTX is 2.5 times higher than PTX. Then, the plasma
The mice were injected with PTX (20 mg kgÀ1 calculated as PTX) concentrations of NO/PTX and PTX went to basically the same after
or NO/PTX (50 mg kgÀ1 calculated as PTX) solution in saline via 4 h. The area under the plasma concentration–time curve (AUC0–t
)
the tail vein. Plasma and tumor were collected at 0, 0.05, 0.5, 1, of NO/PTX was 1.47 folds higher than that of PTX.
2, 3, 4, 8, 12 and 24 h. At each sampling time point, 3 to 4 mice
Paclitaxel concentrations in tumors and various tissues were
were anesthetized with ether, and blood was collected via illustrated in Fig. 6B and C. After i.v. administration, paclitaxel
cardiac puncture from every mouse. These mice were sacrificed was widely distributed into most tissues. Among them, the
and all tumors were also collected. After centrifugation, about highest paclitaxel concentration was obviously found in the
100 mL plasma from each mouse was collected and frozen. The tumor. The paclitaxel concentration of the NO/PTX group in
concentrations of PTX in plasma and in the tumor were the tumor was higher than the PTX group at all time points and
This journal is © The Royal Society of Chemistry and the Centre National de la Recherche Scientifique 2021
New J. Chem., 2021, 45, 13763–13774 | 13767

View Article Online
Paper
NJC
Fig. 5 Inhibition of tumor growth by (A) PTX, Genexols-PM and NO/PTX at 10 and 15 mg kgÀ1; by (B) mPEG-PLA and mPEG-PLA-NO.
Table 2 In vivo tumor growth inhibition (n = 9)
Tumor inhibition
rate %
Group
Dosage mg kgÀ1
Number of animals (n)
Body weight (g)
Tumor weight (g)
Control
Saline
PTX
—
—
10
15
10
15
10
15
9
9
9
9
9
9
9
9
34.0 Æ 1.6
35.5 Æ 2.1
34.6 Æ 1.5
34.2 Æ 2.9
34.4 Æ 2.8
33.5 Æ 2.1
33.0 Æ 2.3
33.0 Æ 2.1
—
—
—
1.67 Æ 0.92
1.03 Æ 0.74
0.79 Æ 0.49
1.07 Æ 0.42
0.99 Æ 0.68
0.99 Æ 0.96
0.55 Æ 0.16
39 Æ 44
53 Æ 29
36 Æ 25
41 Æ 40
41 Æ 57
67 Æ 10
PTX
Genexols-PM
Genexols-PM
NO/PTX
NO/PTX
reached the maximum paclitaxel concentration in 3 h. In these these tissues at the same dose. It is indicated that NO/PTX has
organs, paclitaxel concentrations were tested at a low concen- less accumulation in tissues and organs with improved safety.
tration and mostly eliminated after 24 h. Even though we
2.9 Effects on the expression of related protein
observed the levels in all the tissues with NO/PTX were higher
at each time point than PTX, as shown in Fig. 6C, in fact, the
NO/PTX group exhibited a lower concentration of paclitaxel in
P-Glycoprotein (P-gp) is a plasma membrane protein that acts
as an efflux pump for chemotherapeutic agents, associated with
Table 3 Pharmacokinetic parameters observed in mice after i.v. PTX and NO/PTX
Dose (mg kgÀ1
20
)
Tmax (h) Cmax (mg mLÀ1
)
t1/2b (h) AUC(0–t) (mg h mLÀ1
)
AUC(0–N) (mg h mLÀ1
)
CL (L hÀ1 kgÀ1
)
Vd (L kgÀ1
)
PTX
0.05
0.05
71.73 Æ 15.9
105.16 Æ 3.6
1.485
1.705
83.315
123.154
91.309
115.94
0.39
0.23
0.169
0.242
NO/PTX 50
13768
|
New J. Chem., 2021, 45, 13763–13774
This journal is © The Royal Society of Chemistry and the Centre National de la Recherche Scientifique 2021

View Article Online
NJC
Paper
Fig. 6 Time courses of paclitaxel levels in mice plasma (A) and in mice tumor (B) and tissue distribution of paclitaxel (C) after i.v. administration of
20 mg kgÀ1 dose of PTX and 50 mg kgÀ1 dose of NO/PTX. Each point represents the mean Æ S.D. of three mice per time point.
tumor multi-drug resistance.37–39 The good antitumor effects can 0.001 or 0.01 mg mLÀ1 (Fig. 8A).42–44 The result showed that the
be achieved by inhibiting the function of P-gp or suppressing its NO/PTX group had elevated apoptosis levels, while the control
expression. It was reported that NO donor mainly affected cellular group mitigated apoptosis (P o 0.05). The results manifested
P-gp activity by suppressing the expression of P-gp.34,40,41 To that compared with the PTX group, the level of Cleaved-
determine whether NO/PTX was suppressing P-gp expression, we Caspase-3 and Bax in PTX and NO/PTX groups were obviously
incubated HCT116, SW480, HT29, SGC-7901 and MCF-7 cells with elevated, while the level of Bcl-2 was significantly decreased in
PTX or NO/PTX and utilized western blot analysis to determine the the NO/PTX group.
expression level of P-gp. Compared with the control and PTX
Epithelial–mesenchymal transition (EMT) was considered to
groups, NO/PTX significantly decreased P-gp expression in be an important mechanism for cancer cell migration and
HCT116, SW480, HT29, SGC-7901 and MCF-7 cell lines (Fig. 7A). invasion.45–47 Changes in EMT and invasive properties in vitro
Using HCT116 as a high response cell line, two doses of PTX and could be used to hypothesize the metastatic potential of cancer
NO/PTX were tested for suppression of P-gp. When the dose of PTX cells in vivo. To further investigate the inhibitory effect of
increased, the relative P-gp expression increased as well. On the NO/PTX on the migration and invasion of HCT116 cells,
contrary, the expression of P-gp was declined, in accordance with western blotting was used to examine the expression levels of
the increased amount of NO/PTX (Fig. 7B), which was consistent EMT-related markers. Compared with the PTX group,
with the enhanced antitumor effect of NO/PTX on HCT116.
the expression levels of ZO-1 protein in HCT116 cells were
The levels of apoptosis-related proteins Bax, Bcl-2 and significantly increased, whereas the expression levels of
cleaved caspase-3 were also detected by western blot in interstitial markers vimentin were significantly reduced
HCT116 cell line with the treatment with PTX or NO/PTX at (P o 0.05; Fig. 8B).48,49 Class III b-tubulin counteracts the
This journal is © The Royal Society of Chemistry and the Centre National de la Recherche Scientifique 2021
New J. Chem., 2021, 45, 13763–13774 | 13769

View Article Online
Paper
NJC
Fig. 7 NO/PTX suppressed P-gp expression in five cell lines (A). NO/PTX suppressed P-gp expression in HCT116 at 0.001 and 0.01 mg mLÀ1 (B).
Fig. 8 NO/PTX elevated the level of Bax, Bax/Bcl-2 and cleaved caspase-3 and declined the level of Bcl-2 in HCT116 cell (A). NO/PTX elevated the level
of ZO-1 and declined the level of interstitial markers vimentin and III b-tubulin in HCT116 cell (B).
ability of paclitaxel to inhibit cell migration.50,51 Compared micelle-based drug delivery system for cancer therapy. We eval-
with the PTX group, the expression levels of Class III b-tubulin uated its cytotoxicity against five cancer cell lines (HCT116,
protein in the NO/PTX group were significantly reduced SW480, HT29, SCG7901 and MCF-7). The result showed that
(P o 0.05). The results suggested that NO/PTX could reverse NO/PTX was more competent in anti-proliferative activity against
EMT in HCT116 cells.
gastroenteric cancer cell lines such as HCT116, SW480, SGC-7901
and less competent toward HT29 than PTX at the same concen-
trations. In vivo biological activity results indicated that NO/PTX
had advantages over commercially available PTX injections in
terms of reduced toxicity and improvement of antitumor efficacy.
3. Conclusion
In this study, we demonstrated that NO/PTX could release func- Western blot analysis in HCT116 cells manifested that NO/PTX
tional NO and chemotherapeutic drug paclitaxel, as a polymeric suppressed P-gp expression, elevated apoptosis-related proteins
13770
|
New J. Chem., 2021, 45, 13763–13774
This journal is © The Royal Society of Chemistry and the Centre National de la Recherche Scientifique 2021

View Article Online
NJC
Paper
Cleaved-Caspase-3 and Bax and decreased apoptosis-related pro- (1 mL) and DMAP (146 mg) were added. A solution of anhydride
teins Bcl-2. NO/PTX inhibited cell migration in the wound scratch in CH2Cl2 (15 mL) was added to the mixture at 0 1C. The
assay in HCT116 cells, and acted on epithelial–mesenchymal resulting reaction mixture was stirred at this temperature for
transition (EMT) as well to suppress the migration and invasion 2 h, maintained at room temperature for 36 h, added to cold
of cancer cells. Western blot analysis also showed NO/PTX ethanol. The precipitate was collected by filtration and dried to
increased levels of EMT-related markers protein ZO-1, whereas afford the mPEG-PLA-NO polymer (12.8 g). The structure of the
1
the expression levels of interstitial markers vimentin were polymer was confirmed by H NMR.
significantly reduced. Hence, NO/PTX could be considered as The polymer mPEG-PLA-NO (50 mg) and surfactant HS15
a promising agent against colon cancer with the merits of (50 mg) were dissolved in ethanol (5 mL). PTX (10 mg) was
lowering toxicity, enhanced solubility and antitumor activity.
dissolved completely in the solution after stirring. Mannitol
solution (5%, 1.9 mL) was added to form the aqueous micelle.
The clear solution was lyophilized to afford the micellar PTX
(NO/PTX).
The entrapment efficiency (EE%) of NO/PTX was determined
using an ultrafiltration method by separating the non-entrapped
4. Experimental section
4.1 Material and instruments
mPEG2000 (MW: 2220) and DL-lactide were purchased
from Advanced Polymer Materials Inc. (Montreal, Canada).
Paclitaxel was purchased from Sigma-Aldrich (St. Louis, MO,
USA). Genexols-PM was purchased from Samyang Biopharmaceu-
ticals Corp. (Daejeon, South Korea). Paclitaxel injection was
obtained from Sichuan Sunnyhope Pharmaceutical Co., Ltd
(Chengdu, China). Chemical reagents were obtained from com-
mercial suppliers and used without further purification. 1H-
NMR and 13C-NMR spectra were recorded on a Bruker AVANCE
III HD 600 (600 Hz) spectrometer. Chemical shifts are reported
in parts per million (ppm) downfield relative to tetramethylsi-
lane as an internal standard. HPLC Analysis was performed on a
Waters Acquitys Arct with 2998 PDA detector. The morphology
of NO/PTX nanoparticles was examined using transmission
electron microscopy (TEM) (FEI, Tecnai G2 F20, USA). Its size
diameter was measured using a Zetasizer Nano apparatus (ZS
ZEN3690, Malvern, UK).
KM mice (4–6 weeks, 18–22 g) were provided by Hunan SJA
Laboratory Animals (Hunan, China). The animal experiment was
performed in accordance with China SAC/TC 281 Laboratory
animal-Guideline for ethical review of animal welfare (GB/T
35892-2018) and was approved by the Experimental Animal
Ethics Committee of Jiangxi University of Traditional Chinese
Medicine (JZLLSC2019-0170, Nanchang, China).
drug from nanoparticles. EE% = (Ctotal À Cfree)/Ctotal
.
4.3 NO-releasing test
NO was measured by the Griess reaction. Briefly, NaNO2 at
different concentrations was prepared for the standard curve.
Nitroglycerin (0.00166 mM) and NO/PTX (0.005 mM) were
incubated in an L-cysteine environment and saline was used
as a control, after 1, 2, 3, 4, 6, 8, 10 h, taken 1 mL incubated
solution with 1 mL Griess reagent for 10 min. The resulting
absorbance values at 540 nm were determined using a microplate
reader. The duration of NO release and the highest NO-release
rate as essential parameters were calculated.
4.4 Acute toxicity
Acute toxicity for mPEG-PLA, mPEG-PLA-NO, PTX and NO/PTX
was investigated in healthy male SPF KM mice. 5–8 mice were used
per group and single i.v. doses of 1500, 2000 and 3200 mg kgÀ1
mPEG-PLA, 1500, 2000, 3000 and 3200 mg kgÀ1 mPEG-PLA-NO,
20, 30, 36.3, 38.84, 40.55, 42.3 and 45 mg kgÀ1 PTX. 60, 80, 120,
141.72 and 160 mg kgÀ1 NO/PTX, and saline as a control was
injected through the tail vein. The animals were euthanatized
following the animal (Control of Experiments) ordinance of laws of
China, after the 7 day treatment period, and the number of
mice surviving was recorded. The median lethal dose (LD50
)
and the maximum non-lethal dose (MNLD) were determined by
Graph Pad 6.
4.2 Micelle preparation and characterization
To a vacuum dried mPEG2000 (30.0 g) at 130 1C in the flask was
added DL-lactide (30.6 g) and tin(II) octoate (121 mg). The
4.5 In vitro cytotoxicity
mixture was sealed and stirred at 130 1C for 18 h. After cooling Human colon cancer (CRC) cell lines HCT116, SW480 and
down to room temperature, the crude product was dissolved in HT29, human gastric cancer cells SGC-7901, human breast
CH2Cl2 (50 mL) and cold absolute ethanol (300 mL) was added cancer cell line MCF-7 were used in vitro cytotoxicity study.
to precipitate. The solid was filtered and dried in a vacuum to The growth-inhibitory effect of PTX and NO/PTX were assessed
afford the mPEG-PLA polymer (56.2 g).
based on 10-fold dilutions ranging from 0.0001 up to
The NO-donating furoxan derivative, synthesized according 10 mg mLÀ1 using MTT assay. Briefly, the cells were counted
to our previous study, was coupled with mPEG-PLA polymer via and plated in 96-well plates at a density of 8 Â 103 cells per well
mixed anhydride. Briefly, to the solution of furoxan compound for 12 h and then treated with each drug concentration of PTX
(3.0 g) in EtOAc (30 mL) were added Et3N (1.73 mL) and pivaloyl or NO/PTX in triplicate. Following 48 h of continuous drug expo-
chloride (1.11 mL) at À10 1C. The reaction mixture was stirred sure, the MTT solution (20 mL, 5.0 mg mLÀ1) was poured into each
at 0 1C for 2 h and filtered to remove undissolved salt. The well. After 4 h incubation, the environment containing unreacted
filtrate was evaporated to give a liquid as mixed anhydride. To MTT was discarded from the wells and then DMSO (150 mL) was
the solution of mPEG-PLA (12.6 g) in CH2Cl2 (63 mL), pyridine added to each well. The OD at 570 nm was measured using a
This journal is © The Royal Society of Chemistry and the Centre National de la Recherche Scientifique 2021
New J. Chem., 2021, 45, 13763–13774 | 13771

View Article Online
Paper
NJC
microplate reader (Spectra MAX I3, USA). All experiments were concentration at different times was expressed as mean Æ stan-
performed independently at least three times. SPSS 13.0 software dard deviation (S.D.), and the mean concentration–time curves
was used to analyze the 50% inhibitory concentration (IC50).
were plotted. The maximum plasma or tumor concentration (Cmax)
and the time to reach it was observed directly from the concen-
tration–time curves. The area under the plasma concentration–
4.6 In vitro wound-healing assay
Wound healing assays were used to evaluate the motility and time curve from zero to the time of the final measurable sample
metastatic potential of HTC116 cells. Briefly, cells were seeded (AUC0–t) was calculated using the linear-trapezoidal rule up to the
in 6-well plates at a density of 5 Â 105 cells per well, After 24 h, a last sampling point with the detectable level (C).
200 mL pipette tip at a consistent width was used to wound the
Plasma and tumor samples were pretreated with the liquid–
monolayers. After scratching, the dead cells were removed and liquid extraction method as the plasma, and the ethyl acetate–
washed with PBS three times, Subsequently, PTX and NO/PTX dichloromethane–acetonitrile (4 :1 : 1) was the extraction solvent.
at concentrations of 0.01 and 0.1 mg mLÀ1, respectively, serum- HPLC for the analysis of PTX and docetaxel (internal standard) in
free medium were added. Images in different groups were plasma and tumor was performed on a Waters ACQuity Arc
captured at 0, 12 and 24 h using a Leica DMI3008 inverted system (Milford, MA, USA) with the Empower 3 software (Waters)
microscope. Image J software was used to calculate the wound and a PDA detector (2998, Waters). The analytes were separated
healing area.
on a hyersil ODS2 column (4.6 Â 250 mm, 5.0 mm; Guangzhou,
China) with a wavelength of 227 nm. The analytes were eluted by
acetonitrile: water (40 : 60, v/v, plasma) and (42 : 58, v/v, tumor).
The samples from the heart, liver, spleen, lungs and kidneys
4.7 In vivo tumor growth inhibition
Mice (4–6 weeks,18–22 g) were implanted subcutaneously into
the right flank with 200 mL of cell suspension containing were also pre-treated by liquid–liquid extraction with ethyl acetate–
2 Â 106 H22 cells (in PBS buffer). When the tumor had reached dichloromethane–acetonitrile (4 : 1 : 1) as the extraction solvent.
a volume of 50–150 mm3 treatments were started and the day was
A Shimadzu LC-VP HPLC system (Kyoto, Japan) consisting of
designated on day zero. On day 0, tumor-bearing mice were an LC-30AD pump and a SIL-30AC autosampler was used for
randomly assigned to 7 groups with 12 mice in each group. Treated the separation of PTX and docetaxel on a Welch Ultimate
via the tail vein injection with 10 and 15 mg kgÀ1 PTX, 10 and UHPLC AQ-C18 column (100 Â 2.1 mm, 1.7 mm; Shanghai,
15 mg kgÀ1 Genexols-PM, 10 and 15 mg kgÀ1 NO/PTX and saline as China). The mobile phase was a mixture of 0.1% formic acid in
a control on days 0, 3 and 6. On day 9, mice were euthanized; water (v/v) (mobile phase A) and acetonitrile (mobile phase B).
samples of plasma and tumor were harvested and stored at À80 1C. The gradient elution program was as followed: 55% B, 0 min;
The tumor volume was calculated, as V = W2 Â L/2, where 55–80% B, 0–3.0 min; 55% B, 3.1–5.0 min.
W = longer diameter and L = smaller diameter. In another separate
MS analysis was performed on a Triple Quad 4500 system
study, 6 groups of mice were injected separately with 22.5, from Applied Biosystems (MDS-Sciex, Concord, Canada)
45 mg kgÀ1 PTX, 22.5, 45 and 90 mg kgÀ1 NO/PTX and saline equipped with Turbo V sources and Turbo Ionsprayt interface.
as a control. We also tested the antitumor activity of mPEG-PLA The electrospray ionization (ESI) source was used in a positive
and mPEG-PLA-NO with doses of 300 mg kgÀ1 in the same way. mode, and the mass spectrometric parameters were optimized
as followed: turbo ion spray temperature, 500 1C; ion spray
voltage, 5500 V; curtain gas, nitrogen, 35 L minÀ1; nebulizing
4.8 Pharmacokinetics and tissue distribution
The pharmacokinetics and tissue distribution were approved by the gas, 40 L minÀ1; turbo ion spray gas, 40 L minÀ1; entrance
University Committee on the Use and Care of Animals, Jiangxi potential, 10 V; collision cell exit potential, 14 V. The declustering
University of Traditional Chinese Medicines. The pharmacokinetics potentials for PTX and docetaxel were adjusted to 108 and 95 V,
and tissue distribution were studied in a male KM mouse xenograft respectively. The optimized collision energies for PTX and docetaxel
model established with H22 cells to delineate the disposition of the were 20 and 10 eV, respectively. Quantification was performed
NO/PTX in vivo. The tumors were allowed to grow to about using multiple reactions monitoring (MRM) and the optimized
100 mm3. The mice were injected with PTX solution (prepared with MRM transitions were 854.3 - 286.1 for PTX and 808.6 -
0.9% sodium chloride solution, 20 mg kgÀ1 calculated as PTX) or 226.3 for docetaxel. The quadrupoles Q1 and Q3 were set on the
NO/PTX (50 mg kgÀ1 calculated as PTX) via the tail vein. Plasma and unit resolution. Analyst Softwaret (version 1.6.2) was used to
tumor were collected at 0, 0.05, 0.5, 1, 2, 3, 4, 8, 12 and 24 h. At each process the obtained data.
sampling time point, 3 to 4 mice were anesthetized with ether, and
All methods were validated according to the Guidance for
blood was collected via cardiac puncture from every mouse. Then, Industry, Bioanalytical, Method Validation (US Food and Drug
these mice were sacrificed, and all tumors were also collected. After Administration).
centrifugation, about 100 mL plasma from each mouse was collected
The statistical differences were tested using a one-tailed
and frozen. The concentration of PTX in plasma and tumor was Student t-test at the p o 0.01 or at the p o 0.05 level.
determined by HPLC. The concentration of PTX in the heart, liver,
spleen, lungs and kidneys was determined using HPLC-MS/MS.
4.9 Effects on the expression of related protein
The pharmacokinetic parameters were processed by non- Total proteins were extracted from HCT116 cells after treatment,
compartmental analysis using the DAS 2.0 software package followed by the determination of protein concentration using the
(Chinese Pharmacological Society). The plasma or tumor BCA Protein Assay Kit (ComWin Biotech Co., Ltd, Beijing, China).
13772
|
New J. Chem., 2021, 45, 13763–13774
This journal is © The Royal Society of Chemistry and the Centre National de la Recherche Scientifique 2021

View Article Online
NJC
Paper
Von Hoff and B. Leyland-Jones, J. Clin. Oncol., 1990,
87, 1263.
7 C. Luo, Y. Wang, Q. Chen, X. Han, X. Liu, J. Sun and Z. He,
Mini-Rev. Med. Chem., 2012, 12, 434.
8 X. Li, C. Fan, Z. Xiao, X. Li, C. Fan, Z. Xiao, Y. Zhao,
H. Zhang, J. Sun, Y. Zhuang, X. Wu, J. Shi and Y. Chen, J.
Dai, Biomaterials, 2018, 183, 114.
9 J. Xue, Z. Zhao, L. Zhang, L. Xue, S. Shen, Y. Wen, Z. Wei,
L. Wang, L. Kong, H. Sun, Q. Ping, R. Mo and C. Zhang, Nat.
Nanotechnol., 2017, 127, 692.
Equal amounts of protein (35 mg) were subjected to 7.5% or
12.5% SDS-PAGE and transferred to the PVDF membrane. After
blocking with 5% nonfat milk for 2 h at room temperature, the
membranes were then incubated with primary antibodies against
P-gp, anti-BCL-2, anti-BAX, anti-cleaved caspase 3, anti-vimentin,
anti-ZO-1, anti-Class III b-Tubulin, anti-b-actin, anti-GAPDH with
gentle rotation overnight at 4 1C. Following incubation with
secondary antibody conjugated with horseradish peroxidase
(HRP) for 1 h at room temperature, the immunoreactive bands
were visualized by the enhanced chemiluminescent (ECL) reagent
(ComWin Biotech Co., Ltd., Beijing, China).
10 J. You, R. Shao, X. Wei, S. Gupta and C. Li, Small, 2010, 69, 1022.
11 Y. Yang, Z. Huang, J. Li, Z. Mo, Y. Huang, C. Ma, W.
Wang, X. Pan and C. Wu, Adv. Healthcare Mater., 2019,
8, 1900965.
Author contributions
12 M. M. Hasani-Sadrabadi, V. Karimkhani, F. S. Majedi,
J. J. Van Dersarl, E. Dashtimoghadam, F. Afshar-Taromi,
H. Mirzadeh, A. Bertsch, K. I. Jacob, P. Renaud, F. J. Stadler
and I. Kim, Adv. Mater., 2014, 2619, 3118.
13 Y. Dai, C. Xu, X. Sun and X. Chen, Chem. Soc. Rev., 2017,
4612, 3830.
14 J. R. Upponi, K. Jerajani, D. K. Nagesha, P. Kulkarni, S. Sridhar,
C. Ferris and V. P. Torchilin, Biomaterials, 2018, 170, 26.
15 T. Chen, L. Tu, G. Wang, N. Qi, W. Wu, W. Zhang and
J. Feng, Int. J. Pharm., 2020, 578, 119105.
Huilan Li: data curation, formal analysis, investigation, writing –
original draft. Yuanying Fang: conceptualization, data curation,
writing – review & editing, funding acquisition. Xiang Li:
methodology, investigation. Liangxing Tu: formal analysis.
Guoliang Xu: project administration. Yi Jin: conceptualization,
writing – review & editing, Ronghua Liu: investigation. Zunhua
Yang: conceptualization, formal analysis, funding acquisition,
writing – review & editing.
16 K. Luo, S. Yin, R. Zhang, H. Yu, G. Wang and J. Li, Int.
J. Pharm., 2020, 589, 119870.
Conflicts of interest
The authors declare no conflicts of interest.
17 K. Hadinoto, A. Sundaresan and W. S. Cheow, Eur. J. Pharm.
Biopharm., 2013, 85, 427.
18 K. Y. Choi, K. H. Min, H. Y. Yoon, K. Kim, J. H. Park, I. C.
Kwon, K. Choi and S. Y. Jeong, Biomaterials, 2011, 32, 1880.
19 S. C. Kim, D. W. Kim, Y. H. Shim, J. S. Bang, H. S. Oh,
S. Wan Kim and M. H. Seo, J. Controlled Release, 2001,
72, 191.
20 M. Studenovsky, L. Sivak, O. Sedlacek, R. Konefal, V. Horkova,
T. Etrych, M. Kovar, B. Rihova and M. Sirova, J. Controlled
Release, 2018, 269, 214.
Acknowledgements
This study was supported by Jiangxi Provincial Department of
Science and Technology (20192ACB21012), the Education
Department of Jiangxi Province on Science and Technology
Project Foundation (GJJ201211), Jiangxi University of Traditional
Chinese Medicine (JXSYLXK-ZHYAO013) and Nanchang Innovative
Talent Team (No. [2018]274).
21 P. N. Coneski and M. H. Schoenfisch, Chem. Soc. Rev., 2012,
4110, 3753.
22 B. Bonavida and H. Garban, Redox Biol., 2015, 6, 486.
23 S. Waheed, R. Cheng, Y. Casablanca, G. Maxwell, D. Wink
and V. Syed, Molecules, 2019, 24, 3722.
24 V. Somasundaram, D. Basudhar, G. Bharadwaj, J. H. No,
L. A. Ridnour, R. Y. S. Cheng, M. Fujita, D. D. Thomas, S. K.
Anderson, D. W. McVicar and D. A. Wink, Antioxid. Redox
Signaling, 2019, 308, 1124.
References
1 T. S. Mok, Y. L. Wu, S. Thongprasert, C. H. Yang, D. T. Chu,
N. Saijo, P. Sunpaweravong, B. Han, B. Margono, Y. Ichinose,
Y. Nishiwaki, Y. Ohe, J. J. Yang, B. Chewaskulyong, H. Jiang,
E. L. Duffield, C. L. Watkins, A. A. Armour and M. Fukuoka,
N. Engl. J. Med., 2009, 36110, 947.
25 W. Xu, L. Liu, I. Charles and S. Moncada, Nat. Cell Biol.,
2004, 611, 1129.
26 J. Kim, B. Yung, W. J. Kim and X. Chen, J. Controlled Release,
2017, 263, 223.
27 Y. C. Sung, P. R. Jin, L. A. Chu, F. F. Hsu, M. R. Wang,
C. C. Chang, S. J. Chiou, J. T. Qiu, D. Y. Gao, C. C. Lin, Y. S.
Chen, Y. C. Hsu, J. Wang, F. N. Wang, P. L. Yu, A. S. Chiang,
A. Y. Wu, J. J. Ko, C. P. Lai, T. T. Lu and Y. Chen, Nat.
Nanotechnol., 2019, 1412, 1160.
2 L. Wang, X. Zhao, F. Yang, W. Wu, M. Wu, Y. Li and
X. Zhang, Int. J. Biol. Macromol., 2019, 138, 207.
3 U. Gupta, S. Sharma, I. Khan, A. Gothwal, A. K. Sharma,
Y. Singh, M. K. Chourasia and V. Kumar, Int. J. Biol. Macro-
mol., 2017, 98, 810.
4 R. T. Liggins, W. L. Hunter and H. M. Burt, J. Pharm. Sci.,
1997, 8612, 1458.
5 M. Markman, A. Kennedy, K. Webster, B. Kulp, G. Peterson
and J. Belinson, J. Clin. Oncol., 2000, 181, 102.
28 B. Bonavida, Redox Biol., 2015, 5, 415.
29 H. Yasuda., Nitric oxide, 2008, 192, 205.
6 R. B. Weiss, R. C. Donehower, P. H. Wiernik, T. Ohnuma,
R. J. Gralla, D. L. Trump, J. R. Baker, D. A. Van Echo, D. D.
This journal is © The Royal Society of Chemistry and the Centre National de la Recherche Scientifique 2021
New J. Chem., 2021, 45, 13763–13774 | 13773

View Article Online
Paper
NJC
30 A. W. Carpenter and M. H. Schoenfisch, Chem. Soc. Rev., 43 J. A. Keeble and A. P. Gilmore, Cell Res., 2007, 1712, 97.
2012, 4110, 3742. 44 N. Van Opdenbosch and M. Lamkanfi, Immunity, 2019, 506, 1352.
31 V. Ciccone, M. Monti, E. Monzani, L. Casella and L. Morbidelli, 45 M. A. Nieto, R. Y. Huang, R. A. Jackson and J. P. Thiery, Cell,
Oncotarget, 2018, 917, 13353. 2016, 1661, 21.
32 Q. Li, P. Zou, J. Sun and L. Chen, Eur. J. Med. Chem., 2018, 46 O. D. Persa and C. M. Niessen, Nat. Cell Biol., 2019, 213, 299.
143, 732.
47 J. Yang, P. Antin, G. Berx, C. Blanpain, T. Brabletz, M. Bronner,
K. Campbell, A. Cano, J. Casanova, G. Christofori, S. Dedhar,
33 S. Kudo and Y. Nagasaki, J. Controlled Release, 2015, 217, 256.
34 M. F. Chung, H. Y. Liu, K. J. Lin, W. T. Chia and H. W. Sung,
Angew. Chem., Int. Ed., 2015, 5434, 9890.
35 S. Sindhwani, A. M. Syed, J. Ngai, B. R. Kingston, L. Maiorino,
J. Rothschild, P. MacMillan, Y. Zhang, N. U. Rajesh, T. Hoang,
J. L. Y. Wu, S. Wilhelm, A. Zilman, S. Gadde, A. Sulaiman,
B. Ouyang, Z. Lin, L. Wang, M. Egeblad and W. C. W. Chan,
Nat. Mater., 2020, 195, 566.
36 Y. Fang, R. Wang, M. He, H. Huang, Q. Wang, Z. Yang, Y. Li,
S. Yang and Y. Jin, Bioorg. Med. Chem. Lett., 2017, 27, 98.
37 R. W. Robey, K. M. Pluchino, M. D. Hall, A. T. Fojo, S. E. Bates
and M. M. Gottesman, Nat. Rev. Cancer, 2018, 18, 452.
´
R. Derynck, H. L. Ford, J. Fuxe, A. Garcıa de Herreros, G. J.
Goodall, A. K. Hadjantonakis, R. J. Y. Huang, C. Kalcheim,
R. Kalluri, Y. Kang, Y. Khew-Goodall, H. Levine, J. Liu,
´
G. D. Longmore, S. A. Mani, J. Massague, R. Mayor,
D. McClay, K. E. Mostov, D. F. Newgreen, M. A. Nieto,
A. Puisieux, R. Runyan, P. Savagner, B. Stanger, M. P.
Stemmler, Y. Takahashi, M. Takeichi, E. Theveneau, J. P.
Thiery, E. W. Thompson, R. A. Weinberg, E. D. Williams,
J. Xing, B. P. Zhou and G. Sheng, EMT International Association
(TEMTIA), Nat. Rev. Mol. Cell Biol., 2020, 216, 341.
48 W. Saentaweesuk, N. Araki, K. Vaeteewoottacharn, A. Silsirivanit,
W. Seubwai, C. Talabnin, K. Muisuk, B. Sripa, S. Wongkham,
S. Okada and C. Wongkham, Oncol. Res., 2018, 264, 605.
49 A. E. Patteson, K. Pogoda, F. J. Byfield, K. Mandal,
Z. Ostrowska-Podhorodecka, E. E. Charrier, P. A. Galie,
P. Deptuła, R. Bucki, C. A. McCulloch and P. A. Janmey,
Small, 2019, 15, e1903180.
ˇ´
38 S. Dallavalle, V. Dobricic, L. Lazzarato, E. Gazzano,
M. Machuqueiro, I. Pajeva, I. Tsakovska, N. Zidar and
R. Fruttero, Drug Resist. Updates, 2020, 50, 100682.
39 A. M. Pasello, A. M. Giudice and K. Scotlandi, Semin. Cancer
Biol., 2020, 60, 57.
40 H. Zhang, H. Xu, C. Ashby, Y. Assaraf, Z. Chen and H. M.
Liu, Med. Res. Rev., 2021, 41, 525.
50 A. Ganguly, H. Yang and F. Cabral, Oncotarget, 2011, 25, 368.
41 B. K. Sinha, C. D. Bortner, R. P. Mason and R. E. Cannon, 51 K. Sobierajska, K. Wieczorek, W. M. Ciszewski, I. Sacewicz-
Biochim. Biophys. Acta, Gen. Subj., 2018, 186212, 2806.
42 R. Singh, A. Letai and K. Sarosiek, Nat. Rev. Mol. Cell Biol.,
2019, 20, 175.
Hofman, M. E. Wawro, M. Wiktorska, J. Boncela, I. Papiewska-
Pajak, P. Kwasniak, E. Wyroba, C. S. Cierniewski and
J. Niewiarowska, Biochim. Biophys. Acta, 2016, 18639, 2221.
13774
|
New J. Chem., 2021, 45, 13763–13774
This journal is © The Royal Society of Chemistry and the Centre National de la Recherche Scientifique 2021