Van der Waals force-driven indomethacin-ss-paclitaxel nanodrugs for reversing multidrug resistance and enhancing NSCLC therapy

Article abstract of DOI:10.1016/j.ijpharm.2021.120691

The high expression of multidrug resistance-associated protein 1 (MRP1) in cancer cells caused serious multidrug resistance (MDR), which limited the effectiveness of paclitaxel (PTX) in non-small cell lung cancer (NSCLC) chemotherapy. Indomethacin (IND),

Full text of DOI:10.1016/j.ijpharm.2021.120691

International Journal of Pharmaceutics 603 (2021) 120691
Contents lists available at ScienceDirect
International Journal of Pharmaceutics
journal homepage: www.elsevier.com/locate/ijpharm
Van der Waals force-driven indomethacin-ss-paclitaxel nanodrugs for
reversing multidrug resistance and enhancing NSCLC therapy
Wenbo Kang, Yuanhui Ji*, Yu Cheng
Jiangsu Province Hi-Tech Key Laboratory for Biomedical Research, School of Chemistry and Chemical Engineering, Southeast University, Nanjing 211189, PR China
A R T I C L E I N F O
A B S T R A C T
Keywords:
The high expression of multidrug resistance-associated protein 1 (MRP1) in cancer cells caused serious multidrug
resistance (MDR), which limited the effectiveness of paclitaxel (PTX) in non-small cell lung cancer (NSCLC)
chemotherapy. Indomethacin (IND), a kind of non-steroidal anti-inflammatory drugs (NSAIDs), which has been
confirmed to be a potential MRP1 inhibitor. Taking into account the advantages of old drugs without extra
controversial biosafety issue, in this manuscript, the disulfide bond (-S-S-) was employed for connecting IND and
PTX to construct conjugate IND-S-S-PTX, which was further self-assembled and formed nanodrug (IND-S-S-PTX
NPs). The particle size of IND-S-S-PTX NPs was ~160 nm with a narrow PDI value of 0.099, which distributed
well in water and also exhibited a stable characteristic. Moreover, due to the existence of disulfide bond, the NPs
were sensitive to the high level of glutathione (GSH) in tumor microenvironment. Molecular dynamics (MD)
simulation presented the process of self-assembly in detail. Density functional theory (DFT) calculations revealed
that the main driving force in self-assembly process was originated from the van der waals force. In addition, this
carrier-free nano drug delivery systems (nDDs) could reverse the MDR by downregulating the expression of
MRP1 protein in A549/taxol.
Self-assembled conjugates
Multidrug resistance-associated protein 1
Indomethacin
Paclitaxel
Nanomedicine
1. Introduction
approved by FDA), with a mean diameter of 130 nm, has shown superb
therapeutic index and greater rapid clearance than solvent-based (sb-)
Cancer has been regarded as an urgent public health issue world-
wide. In America, lung cancer was responsible for a quarter of cancer
deaths and had the highest estimated mortality rate in 2020. Meanwhile,
the 5-year relative survival rate of lung cancer was barely 19%, which is
just higher than that of pancreatic cancer (Siegel et al., 2020). Unfor-
tunately, the typical type of lung cancer belongs to NSCLC, accounting
for almost 85% (Duan et al., 2019). First-line chemotherapy was still the
priority choice for NSCLC treatment (NSCLC Meta-analysis Collabora-
tive Group, 2014). Paclitaxel (PTX), a tubulin inhibitor with excellent
anti-tumor potential, which has been applied to some cancer therapies
paclitaxel (Yardley, 2013). Encouraged by the promising performance of
nano-scaled medicine, a variety of multifunctional, novel and greatly
potential nano drug delivery systems (nDDs) has been constructed over
the past few decades. Moreover, more and more nDDs have presented
the characteristics of well-defined structure (Wang et al., 2016), high
drug loading (Zhang et al., 2019), stimulating response to tumor
microenvironment (Cheng and Ji, 2020; Wang et al., 2019a), controlled
drug release (Jin et al., 2019; Zhen et al., 2018), prolonged blood cir-
culation and synergistic treatment (Cheng et al., 2020; Yang et al.,
2018). For example, inorganic nDDs - BP-DcF@sPL, which was based on
black phosphorus, achieved the effect of chemotherapy (apoptosis/ne-
crosis), photothermal therapy (local heat and reactive oxygen species
(ROS) generation) and synergistic immunotherapy by increasing the
secretion of interferon (IFN)-γ and promoting dendritic cell (DC)
maturity (Ou et al., 2018). Ang-3I-NM@(siPLK1 + siVEGFR2), a kind of
Polymeric nDDs, could overcome the blood–brain barrier and showed
massive tumor accumulation and unique ROS responsiveness (Zheng
et al., 2019). PTX-LH2(M) of peptide nDDs possessed cell penetrating
´
(such as breast cancer (Dieras et al., 2020; Tolaney et al., 2015), lung
cancer (Herbst et al., 2018), etc.). However, poor aqueous solubility and
multidrug resistance (MDR) limited its clinical application and chemo-
therapeutic efficacy (Szakacs et al., 2014). In addition, there are non-
negligible side effects including peripheral neuropathy (Hertz et al.,
2018), cardiotoxicity (Herrmann, 2020), nephrotoxicity (Mitin et al.,
2013), serious hemolysis effect (Namgung et al., 2014), etc. Formed by
high-pressure homogenization, Albumin-bound paclitaxel (Abraxane®,
* Corresponding author.
E-mail address: yuanhui.ji@seu.edu.cn (Y. Ji).
https://doi.org/10.1016/j.ijpharm.2021.120691
Received 23 January 2021; Received in revised form 12 April 2021; Accepted 4 May 2021
Available online 7 May 2021
0378-5173/© 2021 Elsevier B.V. All rights reserved.

W. Kang et al.
International Journal of Pharmaceutics 603 (2021) 120691
activity and showed great in-vivo efficacy (Nam et al., 2020). Clofar-
abine - Raltitrexed (CA:RT) of carrier-free nDDs exhibited a high drug
loading, which obtained superior bioavailability, therapeutic effect and
extremely reduced side effects in comparison to free drugs (Wang et al.,
2018). In order to avoid the controversial biosafety issue of carrier
materials, carrier-free nDDs was constructed for NSCLC therapy in this
work, which possesses the nice performances of stability, appropriate
solubility, sensitive release, well-defined structure and synergistic
treatment.
excellent potential to diminish several intractable problems of PTX in
clinical usage, and it contributes to synergetic NSCLCL therapy as a part
of carrier-free nDDs that we constructed.
Considering the synergistic chemotherapy effect of IND, the superior
toxicity towards tumor of PTX and the multifunctional feature of nDDs,
novel carrier-free nDDs of indomethacin-S-S-paclitaxel (IND-S-S-PTX)
was designed and synthesized, where PTX and IND were bridged by a
disulfide bond. The prepared nanoparticles (NPs) could be sensitive to
the tumor microenvironment of high expression of GSH and realized
stimulus release. NPs also overcame the poor solubility of PTX and IND.
MD simulation was employed to describe the self-assembly process of
NPs in detail. Meanwhile, due to the downregulation of intracellular
MRP1, A549/taxol showed more sensitivity towards IND-S-S-PTX NPs.
Nano precipitation has been ubiquitous applied to prepare self-
assembly nanoparticles (Xing et al., 2019). Even though many com-
pounds or complicated materials have been reported to be successfully
self-assembled into nanoparticles, it remained a great challenge for
describing or explaining the mechanisms of self-assembly process in
detail (Ianiro et al., 2019; Kim et al., 2017). However, molecular
simulation could provide a unique angle to explore the mechanism of
self-assembly process (Shao et al., 2020; Tavakkoli et al., 2016). Firstly,
molecular dynamics (MD) simulations can visualize the self-assembly
process in a vivid way by simplifying the model (Eslami et al., 2019b;
Jain et al., 2019). Furthermore, non-bonding forces in the system, such
as hydrogen bonding, electrostatic interactions, van der Waals forces
2. Materials and methods
2.1. Materials and reagent
N,N-dicyclohexylcarbodiimide (DCC), paclitaxel, dimethyl sulfoxide
(DMSO), 4-dimethylaminopyridine (DMAP), indomethacin, 3,3′-
dithiodipropionic acid and coumarin-6 were purchased from Tianjin
Heowns Biochemical Technology Co., Ltd. (Tianjin, China). Methanol
(CH3OH), phosphate buffer saline (PBS, pH = 7.4), dichloromethane
(DCM) and chloroform (TCM) were provided by Nanjing Wanqing
Chemical Glass Ware & Instrument Co., Ltd. (Nanjing, China). Propi-
dium iodide (PI), FITC-labeled Goat Anti-Rabbit IgG (H + L), MRP1/
ABCC1 Rabbit Polyclonal Antibody, Annexin V-FITC, RPMI-1640 me-
dium, A549 cells, fetal bovine serum and A549/taxol cells were offered
by Beyotime Biotechnology Co., Ltd (Shanghai, China). The others
analytically pure reagents were provided by commercial sources and
could be utilized directly.
and π-π stacking, can be captured dexterously, which has been widely
regarded as driving forces in the self-assembly process (Chen et al.,
2020; He et al., 2020; Shen et al., 2016; Wang et al., 2019b; Xiong et al.,
2020; Zhang et al., 2020b). In addition, molecular simulation can also
predict morphology of self-assembly micelle in specific ways (De Nicola
et al., 2015; Li et al., 2019; Nie et al., 2015). More and more multiscale
self-assembly process will be simulated properly and precisely with the
development of algorithm model (Eslami et al., 2019a; Rolland et al.,
2020; Wu et al., 2020). Therefore, for describing the self-assembly
process of nDDs that we designed, MD simulations were utilized to
visualize the formation of nanoparticles. The density functional calcu-
lations were employed to explore the driving forces.
2.2. Synthesis of IND–SS–PTX conjugate
A tricky problem for clinical chemotherapy of paclitaxel is inducing
tumor immunosuppressive environment (TIME) (Chang et al., 2017).
Furthermore, inflammation is often involved in the whole process
(Palucka and Coussens, 2016). Paclitaxel can upregulate inflammatory
ligands and receptors by activating toll-like receptor-4 (TLR4), which
may cause systemic inflammation via inflammatory signaling pathways
(NF-κB, PI3K, MAPK) (Ran, 2015; Volk-Draper et al., 2014). Hence,
there are several inflammatory mediators over secretion such as tumor
The synthetic routes were shown in Scheme 1. Synthesis of IND-OH:
IND (5.0 g, 13.97 mmol), DCC (4.32 g, 20.937 mmol) and DMAP (0.17 g,
1.397 mmol) were respectively dissolved in DCM (25 mL) in the round-
bottom flask. After stirring for 30 min at 0 ◦C, ethylene glycol (1.24 g,
16.755 mmol) was added drop by drop. The mixed system was further
reacted under N2 atmosphere at 0 ◦C. After 36 h, the crude product was
extracted from mixed solution of DCM/water (ratio = 1:1) and subse-
quently purified by column chromatography method (eluent: CH3OH/
DCM = 1/80, V/V, silica gel) to obtain 3.3 g of pure IND-OH (yellow oil).
The yield was 58%. 1H NMR (600 MHz, DMSO) δ 7.67 (s, 2H), 7.64 (s,
2H), 7.05 (d, J = 2.5 Hz, 1H), 6.95 (d, J = 9.0 Hz, 1H), 6.72 (dd, J = 9.0,
2.6 Hz, 1H), 4.81 (s, 1H), 4.08 (d, J = 10.1 Hz, 2H), 3.78 (s, 2H), 3.77 (s,
3H), 3.60 – 3.56 (m, 2H), 2.22 (s, 3H). MS (ESI) m/z for C21H21NClO5
[M + Na]+: 424.0. Synthesis of IND-COOH:⋅10 mL TCM liquid con-
tained DCC (1.337 g, 6.48 mmol) was added quite slowly to the mixed
system of DMAP (60 mg, 0.49 mmol), 3,3′-dithiodipropionic acid (1.41
g, 6.71 mmol) and IND-OH (2 g, 4.986 mmol) under dry N2. After stir-
ring for 12 h, 20 mL TCM contained DCC (0.67 g, 3.24 mmol) was added
and further heated to 65 ◦C. After refluxing for 3 h, crude product was
poured into the separating funnel (500 mL) which contained the mixed
solution of water and DCM (ratio: 1/1, V/V). Finally, column chroma-
tography method was employed to gain the pure IND-COOH (eluent:
CH3OH/DCM = 1/40, V/V, silica gel). The yield was 83% (IND-COOH,
yellow oil, 2.45 g). 1H NMR (600 MHz, DMSO) δ 12.40 (s, 1H), 7.73 –
7.57 (m, 4H), 7.05 (d, J = 2.5 Hz, 1H), 6.94 (d, J = 9.0 Hz, 1H), 6.72 (d,
J = 9.0 Hz, 1H), 4.27 (s, 4H), 3.78 (d, J = 12.7 Hz, 5H), 2.85 (dt, J = 9.7,
6.9 Hz, 4H), 2.67 – 2.55 (m, 4H), 2.23 (s, 3H). HRMS (ESI) for
necrosis factor-α (TNF-α), vascular endothelial growth factor-A (VEGF-
A), interleukin(IL)-1β, IL-6, IL-8 (Coussens et al., 2013; Nguyen et al.,
2017; Shalapour and Karin, 2019). Those circulating inflammatory cy-
tokines will promote the maturation, differentiation and recruitment of
immunosuppressive cells (regulatory T cells (Treg), myeloid-derived
suppressor cells (MDSC), etc.), and further contribute to the TIME
(Garner and de Visser, 2020). Multidrug resistance (MDR) is another
obstacle which extremely restricts the clinical usage of PTX (Vaidya-
nathan et al., 2016). Multidrug resistance-associated protein 1 (MRP1),
a kind of ABC transporter proteins, has tremendous ability to efflux
massive anticancer drugs from cancer cells, which causes the low
accumulation of anticancer drugs and the failing outcome of chemo-
therapy (Robey et al., 2018). Recently, immense evidence suggested that
indomethacin (IND) not only applied to pain relief, fever recovery and
anti-inflammatory but also could synergize antitumor activities. More
importantly, IND was a great candidate for MRP1 inhibition (Lolli et al.,
2019). Several in vivo models have confirmed that IND could down-
regulate the expression of MRP1 protein and increase the intracellular
accumulation of chemotherapy drugs (Lee et al., 2017; Zeng et al.,
2020). Meanwhile, IND greatly improved efficacy on revising TIME and
promoting immune response (Zhang et al., 2019). Extensive research
has shown that IND can inhibit the synthesis of prostaglandin E2 and
increase the polarization of M1 phenotype macrophages, which may
help to rebalance the immunity and inhibit the tumor immune escape
(Martinez-Colon and Moore, 2018). Therefore, indomethacin has
C
27H28ClNO8S2 [M + H]+: 594.10422. Synthesis of IND-S-S-PTX: DCC
(0.725 g, 3.51 mmol) in 5 mL TCM solution was added to 30 mL DCM
solution containing IND-COOH (2.34 g, 3.94 mmol), PTX (2.5 g, 2.93
mmol) and DMAP (35.8 mg, 0.293 mmol) under nitrogen protection.
After stirring at 0 ◦C for 48 h, the crude product was acquired by
2

W. Kang et al.
International Journal of Pharmaceutics 603 (2021) 120691
Scheme 1. Chemistry synthetic route of IND-S-S-PTX. Reagents and conditions: I) DCM, glycol, DCC, DMAP, IND, 0 ◦C, N2, 36 h; II)DCC,DMAP, 3,3′-Dithiodi-
propionic, TCM, reflux, 12 h; III) PTX, DCC, DMAP, DCC, TCM, DCM, 0 ◦C, 48 h.
extracting from the mixed solution of DCM and water (ratio = 1:1).
Then, column chromatography method (eluent: CH3OH/DCM = 1/100,
V/V, silica gel) was utilized to acquire 3.2 g pure IND-S-S-COOH (light
yellow solid). The yield of reaction was 76.5%. 1H NMR (600 MHz,
DMSO) δ 9.19 (d, J = 8.6 Hz, 1H), 7.98 (d, J = 7.1 Hz, 2H), 7.84 (d, J =
7.1 Hz, 2H), 7.74 (t, J = 7.3 Hz, 1H), 7.69 – 7.61 (m, 6H), 7.54 (t, J = 6.1
Hz, 1H), 7.51 – 7.41 (m, 6H), 7.22 – 7.17 (m, 1H), 7.03 (d, J = 2.5 Hz,
1H), 6.93 (d, J = 9.0 Hz, 1H), 6.71 (dd, J = 9.0, 2.5 Hz, 1H), 6.29 (s, 1H),
5.83 (t, J = 8.7 Hz, 1H), 5.57 (s, 1H), 5.42 (d, J = 7.2 Hz, 1H), 5.37 (d, J
= 8.8 Hz, 1H), 4.90 (d, J = 11.0 Hz, 2H), 4.63 (s, 1H), 4.26 (d, J = 3.2
Hz, 4H), 4.14 – 4.09 (m, 1H), 4.01 (dd, J = 16.5, 8.2 Hz, 2H), 3.77 (d, J
= 16.6 Hz, 5H), 3.58 (d, J = 7.2 Hz, 1H), 2.88 (t, J = 6.9 Hz, 2H), 2.80
(dd, J = 14.1, 7.3 Hz, 4H), 2.57 (d, J = 13.9 Hz, 2H), 2.32 (t, J = 11.7 Hz,
1H), 2.23 (d, J = 15.8 Hz, 6H), 2.10 (s, 3H), 1.85 – 1.76 (m, 4H), 1.66 –
1.61 (m, 1H), 1.51 (d, J = 16.7 Hz, 4H), 1.01 (d, J = 17.9 Hz, 6H). HRMS
(ESI) for C74H77ClN2O21S2 [M + Na]+: 1451.40844.
was subsequently minimized to 10 KJ/mol by the steepest descent
method. NVT MD simulation was utilized to simulate the operation for
20 ns to acquire a reasonable initial configuration. With further running
5 ns of NPT MD simulation, the system maintained the state of 300 K and
1.0 atm. Then, 15 ns of NPT MD simulation was performed again to
obtain equilibrium. Another NPT MD simulation of 200 ns was con-
ducted for data analysis with the trajectories stored every 1000 fs. In
order to shorten the calculation time, SAHAKE method was hired to fix
the bond between the system and H atom in the whole MD simulation
process. The leap frog integrator was further employed to solve New-
ton’s equations of motion with a time step of 2 fs, and periodic boundary
conditions were used in the three-dimensional direction. For the non-
bonding interactions in the mixed system, the cutoff distance was set
as 1.4 nm, and the long-range electrostatic interaction was calculated by
PME method. Meanwhile, Berendsen thermostat and Parrinello Rahman
algorithm were utilized to control the temperature and pressure of the
system. The coupling constants were 100 fs and 2000 fs respectively. All
MD simulations were completed by GROMACS 5.1.4.
2.3. Preparation and characterization of IND-S-S-PTX NPs
2.4.2. DFT calculations
IND-S-S-PTX NPs were formed by nano-precipitation method. In
short, 1 mg IND-S-S-PTX was dissolved by 1.5 mL DMSO. Subsequently,
the solution was added to 15 mL PBS drop by drop, stirring while add-
ing. After 2 h, the mixed solution was placed into a dialysis bag (MW =
3.5 KDa) and subsequently dialyzed in deionized water for 48 h at 25 ◦C.
Changing the water every 4 h and using 2 L deionized water for dialysis
each time. Dynamic light scattering (DLS, Malvern Zetasizer Nano-ZS90,
Malvern) was utilized to analyze the average size and the poly dispersity
index (PDI) of nanoparticles in solution. The morphology of nano-
particles was observed by TEM (JEM-2100F, JEOL). Coumarin-6-loaded
IND-S-S-PTX NPs were successful prepared (see Fig. S8) by using the
same method of IND-S-S-PTX NPs.
DFT calculation was performed at M062X-D3/6-311G theoretical
level to optimize the bimolecular IND-S-S-PTX and gain the binding
energy, where D3 represented the Grimme’s empirical dispersion
correction (Zhang et al., 2020a). VMD was used to paint the molecular
surface electrostatic potential. Gaussian 16 program package was
employed to perform all the quantum mechanical calculations.
EBinding = EAB ꢀ EA ꢀ EB + BSSE
(1)
E
AB: Counterpoise corrected energy; EA: total energies of monomer A.
EB: total energies of monomer B; BSSE: basis set superposition error.
2.4. Computational simulations
2.5. In vitro IND-S-S-PTX NPs release
Molecular dynamics (MD) simulations and density functional theory
(DFT) calculations were conducted to explore the self-assembly mech-
anism of IND-S-S-PTX NPs.
The GSH-sensitively accumulative release of PTX and IND from IND-
S-S-PTX NPs were conducted in the release medium of PBS (pH = 7.4)
containing 10% FBS, with or without 10 mM GSH at 37.3 ◦C, under a
shaking at the speed of 120 rpm/min. The dialysis bag (MW = 1000 Da)
containing 1 mL solution of IND-S-S-PTX NPs was put into 250 mL
release media. 1 mL release media was withdrawn and determined at the
selected time. The high-performance liquid chromatography (HPLC,
essential IC-16, Shimadzu) was applied to determine the content of PTX
(mobile phase: methanol/acetonitrile/water = 5/7/8) or IND (mobile
phase: methanol/0.05% phosphoric acid aqueous solution = 70/30, V/
V). During the whole process, the UV detection was set at 227 nm or 320
nm, the flow rate was 1 mL/min and the temperature of C18 column was
2.4.1. Molecular dynamics simulations
In this work, GAFF force field was used to describe IND-S-S-PTX and
water, where the non-bond interaction was depicted by the coulomb
potential and the Lennard-Jones (L-J) potential. The mixed L-J param-
eter was obtained from the Berthelot–Lorentz (L-B) mixing rule (Wang
et al., 2004). Firstly, a cube box was constructed by packmol, which
contained 10 IND-S-S-PTX molecules and 20,000 water molecules. In
order to avoid overlapping of atomic coordinates, the energy of system
3

W. Kang et al.
International Journal of Pharmaceutics 603 (2021) 120691
35 ◦C.
2.11. ROS assay
The intracellular ROS level was determined by 2′,7′-dichlorodihy-
drofluorescein diacetate (DCFH-DA) kit. Briefly speaking, A549 and
A549/taxol cells were cultured and treated under the similar method of
2.6. Cell culture and uptake study
A549 and A549/taxol were cultured with RPMI-1640 medium at
37 ◦C in a humidified incubator consisted of 5% CO2. The culture me-
dium containing 1 × 105 U/L penicillin, 10% fetal bovine serum (FBS)
and 100 mg/L streptomycin.
cell apoptosis study while the dose of the drugs was 5
μM. The cells were
washed (2 mL, 3 times, PBS), stained (0.5 mL, 20
μM, DCFH-DA) and
cultured for another 1 h under the similar condition above. The cells
were washed with PBS solution. Eventually, flow cytometer was hired to
analysis intracellular ROS level by detecting the dichlorofluorescein.
The cellular uptake of drugs was observed by confocal laser scanning
microscope (CLSM). In order to make the IND-S-S-PTX NPs have fluo-
rescence signal, we attempted to load coumarin-6 into IND-S-S-PTX NPs.
The cell nuclei were stained by DAPI. During the whole process, in short,
2 × 104 A549 cells were seeded into 24-well plates and incubated for 24
h. The media was removed and the cells were cultured with coumarin-6
2.12. Statistical analysis
All the experimental data are presented as mean ± SD. SPSS 22.0
software was utilized to determine the statistical significance.
(1 μg/mL) loaded in 40 μM IND-S-S-PTX NPs. After 1 h, the cells were
washed (PBS, 3 times), fixed (paraformaldehyde, 14%, 15 min), stained
and ultimately observed by CLSM.
3. Results
3.1. Synthesis of IND-S-S-PTX conjugate
2.7. In vitro cytotoxicity measurement
In order to make the conjugated drug molecules sensitive to the
tumor microenvironment (TME), bridging disulfide bond as linker might
be an appropriate choice (Sun and Zhong, 2020). Owing to the active
group (carboxyl group) of indomethacin, indomethacin glycol fragment
was subsequently given, which was corresponded to IND-OH. Eventu-
ally, 3,3′-dithiodipropionic acid was inserted as responsive fragment,
which linked two hydroxyl compounds (PTX and IND-OH) and gener-
ated IND-S-S-PTX. All the intermediates were confirmed by 1H NMR and
HRMS. The results were presented in Figs. S1–S6.
The potential cytotoxicity of IND-S-S-PTX NPs, IND and PTX towards
cancer cells were assessed by MTT method. Namely, 2 × 104 A549 and
A549/taxol were respectively seeded into 96-well plates, cultured for 24
h and made the cells adhere to the wall. Then, cancer cells were incu-
bated with IND, PTX and IND-S-S-PTX NPs under the concentrations of
0.125, 0.5, 2, 8 and 32 μM. After 24 h, the culture media was removed
and cancer cells were washed by using PBS.20 μL PBS solutions with 5
mg/mL MTT reagent were added into each well, A549 or A549/taxol
cells were further incubated for another 4 h. the medium was removed
3.2. Characterization of IND-S-S-PTX NPs
and 150 μL DMSO was added. The absorbance of solution was measured
at 570 nm by SpectraMax M2e molecular devices before centrifuging for
10 min. The cell viability was eventually evaluated by the relative ab-
sorption ratio of treatment and control groups. IC50 values were ob-
tained by GraphPad Prism software.
IND-S-S-PTX NPs were prepared by nanoprecipitation which was
universally employed to construct nanoparticles (Cheng et al., 2020).
The mean diameter of IND-S-S-PTX NPs was measured by DLS as 157.4
± 0.68 nm (Fig. 1A), which agreed with the results observed by the TEM
micrograph as shown in Fig. 1B. Therefore, it was speculated that IND-S-
S-PTX NPs could accumulate abundantly into the TME by enhancing
permeation and retention (EPR) effect (Zhang and Ji, 2019). A narrow
PDI value of 0.099 ± 0.01 indicated that IND-S-S-PTX NPs were well
distributed in water. Moreover, it might keep stable in PBS containing
10% FBS under 4 ◦C (Fig. 1C). The critical micelle concentration of IND-
S-S-PTX was 0.0539 mg/L as shown in Fig. 1D.
2.8. Cell apoptosis study
1 × 104 A549 and A549/taxol cells were seeded into 6-well plate and
cultured at the same condition above. The cells were treated with IND-S-
S-PTX NPs, IND and PTX at the concentrations of 40 μM. After 48 h, all
the cancer cells were digested with trypsin and subsequently centrifuged
for 10 min. Annexin V-FITC and PI were used to stain the cells for the
further apoptosis analysis by FACScan flow cytometer.
3.3. Self-assembly mechanism of nanoparticles
Here, MD simulations and DFT calculations were performed to
analyze the mechanism of IND-S-S-PTX self-assembly process. Fig. 2
showed a dynamic snapshot of IND-S-S-PTX self-assembly process at
different times. Firstly, 10 drug molecules were randomly dispersed into
a cube containing 20,000 water by packmol. Initial equilibrium state of
0 ns was acquired by the 20 ns of NVT and NPT simulations. With the
further performance of MD simulation, IND-S-S-PTX gradually gathered
and eventually formed compact clusters at 200 ns. On the other hand,
the solvent-accessible area gradually decreased to 84 nm2, the average
number of hydrogen bonds between IND-S-S-PTX and water decreased
to 116, and the value of ΔGsolv was nearly increased to ꢀ 100 kJ/(mol ×
nm2) as shown in Fig. 3A and B, which indicated that IND-S-S-PTX
spontaneously self-assembled into cluster in water.
2.9. Determination of MRP1 level
The expression of MRP1 in A549 and A549/taxol were estimated by
immunofluorescence. The cancer cells were cultured and treated by
using similar way above. Subsequently, A549 or A549/taxol cells were
incubated with MRP1/ABCC1 Rabbit Polyclonal Antibody for 2 h. IgG
(H + L) (FITC-labeled Goat Anti-Rabbit IgG (H + L)) was employed to
treat the cells at 25 ◦C for 0.5 h. ImageJ.JS software was hired to
quantify the level of MRP1 by the fluorescent intensity. Besides, the cells
were photographed by CLSM.
2.10. Cell-cycle analysis
It was difficult to form stable NPs from the single hydrophobic drugs
by self-assembly process, while the modified amphiphilic drugs were
much easier (Han et al., 2016). This might be due to the complex mo-
lecular interactions. Non-bonding interactions are always deemed as
driving force in whole self-assembly process, for instance, van der Waals
1 × 105 A549 and A549/taxol cells were seeded into 6-well plate and
cultured overnight respectively. Then, the media was replaced and cells
were cultured with 40 μM PTX, IND and IND-S-S-PTX NPs. The cells
were collected, fixed (70% ethanol V/V, ꢀ 20 oC, 12 h) and stained with
PI (50
μ
g/mL, 30 min, 4 ◦C) for the further analysis of cell cycle distri-
force, hydrogen bonding,
π-π stacking, etc. To verify the driving force of
bution by flow cytometry.
self-assembly process, the interaction between molecules was further
4

W. Kang et al.
International Journal of Pharmaceutics 603 (2021) 120691
Fig. 1. (A) The size of IND-S-S-PTX NPs analyzed by DLS. (B) TEM image of IND-S-S-PTX NPs (C) The size change of IND-S-S-PTX NPs in PBS solution (pH = 7.4)
containing 10% FBS, 4 ◦C. (D) The critical micelle concentration of IND-S-S-PTX. The in vitro release study of IND-S-S-PTX NPs. The GSH-sensitively accumulative
release of IND (E) and PTX (F), under the condition of PBS with pH of 7.4 with or without 10 mM GSH.
Fig. 2. Dynamic snapshot of IND-S-S-PTX self-assembly process.
detected by DFT calculation. Radial distribution functions (RDF) were
able to provide the probability distribution of H2O around IND-S-S-PTX
(Zhang et al., 2020a). All oxygen atoms in IND-S-S-PTX and H atoms in
H2O were selected as reference sites to investigate the hydrogen bonds
between IND-S-S-PTX and water. Fig. 3C and 3D showed that the first
peak of O1-H RDF was extremely sharp and high. The value of peak
height was 1.15 and the peak position was 1.78 Å, which meant the
existence of hydrogen bond. Similarly, there were obvious hydrogen
bonding interactions between O2, O3, O3, O4, O5, O6, O7, O8, O9, O10,
O11 and water molecules. It was consequently speculated that there was
strong hydrogen bond interaction between IND-S-S-PTX and water,
which allowed the clusters to be dispersed well in water.
IND-S-S-PTX had obvious negative electrostatic potential characteris-
tics, while the hydroxyl group and carbonyl group containing lone pair
electrons had obvious positive electrostatic potential characteristics.
The extreme value of the electrostatic potential were +50.6 kJ/mol and
ꢀ 51.26 kJ/mol, respectively, which indicated IND-S-S-PTX molecules
might produce strong non-bond interaction between IND-S-S-PTX and
themselves. IGM analysis clarified that the non-bond interaction
belonged to van der Waals force (Fig. 4B). The calculation of binding
energy was ꢀ 123.094 kJ/mol (Table S1), which confirmed that the van
der Waals force was strong. The bimolecular structure was also selected
for ESP analysis. It was found that the clusters formed by IND-S-S-PTX
had non-bond interaction with water molecules too, which was agreed
with the result of RDF analysis.
Electrostatic potential (ESP) analysis can adequately exhibit the
characteristics of molecules, which is an effective means to predict non-
covalent interactions. As observed in Fig. 4A, the aromatic ring region of
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International Journal of Pharmaceutics 603 (2021) 120691
Fig. 3. (A) Solvent accessible surface area of IND-S-S-PTX cluster and the average number of hydrogen bonds between water and IND-S-S-PTX cluster. (B) Solvation
free energy of IND-S-S-PTX nanostructures. (C) Oxygen atom diagram of RDF analysis. (D) RDF analysis of oxygen atom in IND-S-S-PTX.
Fig. 4. (A) The ESP diagram of IND-S-S-PTX and bimolecular (B) Independent gradient model (IGM) analysis between IND-S-S-PTX and IND-S-S-PTX molecules.
3.4. In vitro conjugate degradation and PTX release study
could respond to the TME and this may be due to the degradation of
disulfide bond (-S-S-) (Xue et al., 2016).
It had been reported that the expression of GSH in TME was much
higher than that in normal tissues, which allowed the selective release of
IND-S-S-PTX NPs by the cleavage of the disulfide bond (Zhang and Ji,
2020). As shown in Fig. 1F, PTX was rapidly and massively released at
the first 2 h under the condition of 10 mM GSH (PBS, pH = 7.4), while
the IND was slow-released. Compared with the control group, the
accumulative release of IND and PTX in 48 h were 57.8% and 71.3%
respectively. However, less than 10% PTX and IND were released in a
release medium without GSH. The result of the in-vitro release study
approved the assumption that this carrier-free nDDs of IND-S-S-PTX
3.5. Cellular uptake
To demonstrate the cellular uptake of IND-S-S-PTX NPs, A549 and
A549/taxol cells were respectively incubated with IND-S-S-PTX NPs,
then further imaged by CLSM. Coumarin-6 was a great candidate to
solve the shortcoming of IND-S-S-PTX NPs without inherent fluorescent
signal (Xu et al., 2020). Here, coumarin-6-loaded IND-S-S-PTX NPs
could present green fluorescent characteristics (Fig. 5). The cell nucleus
was stained to blue by DAPI. As shown in Fig. 5A, coumarin-6-loaded
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W. Kang et al.
International Journal of Pharmaceutics 603 (2021) 120691
Fig. 5. Cellular uptake photos of coumarin-6 (1
μg/mL) loaded IND-S-S-PTX NPs by CLSM. (A) 1 h in A549 cells. (B) 3 h in A549 cells. (C) 1 h in A549/taxol cells. (D)
3 h in A549/taxol cells.
IND-S-S-PTX NPs could be quickly taken up by A549 in 1 h. Compared to
Fig. 5B, there was no green fluorescent outside the A549 cells after 3 h.
The same circumstances also appeared for A549/taxol, as exhibited in
Fig. 5C and 5D. Therefore, it was observed that both A549 and A549/
taxol could uptake IND-S-S-PTX NPs easily, smoothly and adequately.
μM). Moreover, the IC50 value of IND-S-S-PTX NPs was close to half of
IND (19.343 ± 1.67 μM). Hence, it was confirmed that IND-S-S-PTX NPs
exhibited the most cytotoxicity and superior antitumor potential in
A549/taxol, as observed in Table 1. Furthermore, the cell viability was
decreased with the increased concentration of drugs. When the con-
centration of drugs was 8 μM, PTX and IND-S-S-PTX NPs showed the
similar suppress cell proliferation in A549 (Fig. 6A). However, the re-
sults changed much in A549/taxol cells under the same concentration of
drugs (Fig. 6B), IND-S-S-PTX NPs showed stronger ability in suppress
cells proliferation, which consisted with the results of IC50 values. In
vitro cytotoxicity assay verified that IND-S-S-PTX NPs showed
outstanding potential in A549/taxol.
3.6. In vitro cytotoxicity assay
In vitro cytotoxicity was a significant evaluation indicator for IND-S-
S-PTX NPs, which reflected the potential antitumor effect. In order to
figure out the bioactivity of IND-S-S-PTX NPs, MTT assay were con-
ducted by using A549/taxol and A549 cells. IND and PTX were
employed as positive controls. IC50 results were presented in Table 1 and
Fig. 6. Compared to the results of antitumor effect, there was no obvi-
ously difference between PTX and IND-S-S-PTX NPs in A549 cells. The
3.7. Cell apoptosis study
The cell apoptosis results were respectively presented in Figs. 7 and
8. The total apoptotic ratio of all groups in A549 were 49.4% (IND-S-S-
PTX NPs), 21.49% (IND) and 65.5% (PTX). IND-S-S-PTX NPs was
slightly inferior to PTX as shown in Fig. 7. However, in A549/taxol cells,
IND-S-S-PTX NPs exhibited greater ability to induce apoptosis. The total
apoptotic ratio of IND-S-S-PTX NPs was 49.0%, which was nearly 5 times
as high as that of PTX (11.50%) and twice as high as that of IND (24.4%).
Hence, it was obvious that IND-S-S-PTX NPs showed more sensitive to
A549/taxol cells, which exhibited the feature of reversing multidrug
resistance.
IC50 value of IND-S-S-PTX NPs was 10.247 ± 0.92
μM which just slightly
higher than PTX but much lower than IND. While for the results in
A549/taxol, the IC50 value of PTX was 29.871 ± 2.52 μM, which was
nearly three times as high as that of IND-S-S-PTX NPs (10.791 ± 0.62
Table 1
Cytotoxicity of IND-S-S-PTX NPs, IND, and PTX towards A549 and A549/taxol.
Groups
IC50 values (
μM)
A549
A549/taxol
IND-S-S-PTX NPs
10.247 ± 0.92
21.646 ± 1.92
6.243 ± 0.45
10.791 ± 0.62
19.343 ± 1.67
29.871 ± 2.52
IND
PTX
7

W. Kang et al.
International Journal of Pharmaceutics 603 (2021) 120691
Fig. 6. Cell viability of (A) A549 or (B) A549/taxol after treated with IND-S-S-PTX NPs, IND and PTX in 24 h.
Fig. 7. Cell apoptosis study of IND-S-S-PTX NPs, IND, and PTX towards A549.
Compared to the control group, G0/G1 phase cells were decreased from
3.8. Cell-cycle analysis
13.82% to 0, and G2/M phase cells also decreased from 36.82% to
28.35%. However, the S phase cells were greatly increased from 49.36%
to 71.56%. In addition, the function mechanisms of IND-S-S-PTX NPs
working on A549/taxol cells might be different from that of PTX. The
percentage of cell cycle arrest in S phase was much higher than G2/M
In order to figure out the effect of IND-S-S-PTX NPs on cell cycle
arrest, flow cytometry was implemented on A549 and A549/taxol cells.
The results were shown in Fig. 9. As observed from Fig. 9, IND-S-S-PTX
NPs might tend to arrest A549/taxol cells in S phase and G2/M phase.
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W. Kang et al.
International Journal of Pharmaceutics 603 (2021) 120691
Fig. 8. A549/taxol apoptosis induced by IND-S-S-PTX NPs, IND, and PTX.
phase. The cell-cycle results of A549 were shown in Fig. S7.
Compared to control group, IND-S-S-PTX NPs could induce more ROS
generation than other group in A549/taxol as observed in Fig. 11.
Meanwhile, there were lower ROS level after treated with IND-S-S-PTX
NPs than that treated with PTX group as found in Fig. S6. Therefore,
combined with the performance of IND-S-S-PTX NPs in apoptosis and
cytotoxicity assay, the high level of ROS might contribute to the in-
duction of cell-cytotoxicity and apoptosis.
3.9. MRP1 level determination
The overexpression of MRP1 protein was associated to MDR. IND had
been proved to be an excellent MRP1 inhibition in A549/taxol cells. The
MRP1 level was analyzed by the fluorescent intensity and the mean
values were acquired from ImagineJ JS software. As shown in Fig. 10A,
MRP1 protein was expressed in both A549 and A549/taxol, but more in
A549/taxol. After treated with IND-S-S-PTX NPs, the expression of
MRP1 was immediately decreased. The extent of reduction decreased
significantly in A549/taxol cells. In order to observe the treatment effect
intuitively, CLSM was employed. The results were presented in Fig. 10B
and C. MRP1 protein was stained to green, while the nucleus was stained
to blue. In A549/taxol, control group showed apparent green fluorescent
sign, which meant the high level of MRP1. After incubated with IND-S-S-
PTX NPs, the green fluorescent nearly disappeared (Fig. 10C). However,
the PTX group remained strong green fluorescent. Hence, it was deduced
that the down regulation of the expression of MRP1 in A549/taxol cells
might be mainly responsible for excellent cell cytotoxicity and induced
superior apoptosis of IND-S-S-PTX NPs.
4. Discussions
Multidrug resistance limits the clinical usage of chemotherapy drug
paclitaxel. A549/taxol expresses high level of MRP1 protein, which
belongs to ABC transporters and associates closely with MDR (Stefan and
Wiese, 2019). Paclitaxel is ingested by A549/taxol while MRP1 protein
pumps it out, which causes the severe MDR. Nevertheless, the results of
in vitro cytotoxicity assay and cell apoptosis study of A549 and A549/
taxol exhibited different features. IND-S-S-PTX NPs were extremely
sensitive to the A549/taxol and had excellent cytotoxicity and superior
ability to induce apoptosis compared to paclitaxel. However, those ad-
vantages vanished in A549, the antitumor effect of IND-S-S-PTX NPs was
nearly close to that of paclitaxel. This might be attributed to the
following three main reasons. Firstly, compared to A549 cells, it main-
tained high level expression of MRP1 in A549/taxol cells. Under this
circumstance, MDR might be the leading factor to restrict the therapy
effect of paclitaxel. However, it was IND in IND-S-S-PTX NPs that could
remarkably down regulate the expression of MRP1 in A549/taxol and
3.10. ROS measurement
The intracellular ROS level of A549 and A549/taxol was detected by
DCF-DA Kit. The results were displayed respectively in Figs. 11 and S6.
9

W. Kang et al.
International Journal of Pharmaceutics 603 (2021) 120691
Fig. 9. The cell-cycle distribution of A549/taxol after incubating with IND-S-S-PTX NPs, IND, and PTX.
Fig. 10. (A) The mean immunofluorescence intensity of MRP1 in A549 or A549/taxol which was calculated by imagineJ JS. The expression of MRP1 in A549 (B) or
A549/taxol (C) after treating with drugs imaged by CLSM.
reduce the paclitaxel outflow. Hence, the IC50 value of IND-S-S-PTX NPs
was about a third of that of PTX and the apoptotic ratio of IND-S-S-PTX
NPs was nearly 5 times as high as that of PTX. Moreover, in A549 cells,
the high expression of MRP1 might not be the foremost obstacle for
treatment. Although IND-S-S-PTX NPs could selectively respond to the
TME with high level of GSH, the accumulated release ratio of PTX was
only approximately 70% in 24 h, the low concentration of effective
drugs might cause the antitumor effect of IND-S-S-PTX NPs barely the
10

W. Kang et al.
International Journal of Pharmaceutics 603 (2021) 120691
Fig. 11. Intracellular ROS level of A549/taxol measured by flow cytometry.
same level as PTX. Secondly, IND-S-S-PTX NPs could induce the more
massive intracellular ROS generation in A549/taxol compared to that in
A549 as observed the Figs. 11 and S6. It had been reported that the high
level of ROS could contribute to the death of cancer cell, which was also
the core strategy of photodynamic therapy (Teh et al., 2020). Finally,
the cell-cycle interference mechanism of IND-S-S-PTX NPs was changed.
A549 or A549/taxol cells after been incubated with IND-S-S-PTX NPs
showed the characteristics of arresting the S and G2/M phase cells,
especially for S phase cells. It was quite different from either IND or PTX.
Although the antitumor effect of IND-S-S-PTX NPs was similar to that
of PTX in A549 cells, carrier-free nDDs in the form of aqueous solution
could improve the solubility of PTX. In addition, IND-S-S-PTX NPs could
sensitively respond to the TME with high-level GSH. The low accumu-
lated release ratio of PTX might have promising performance in
enhancing the drug circulation. The suitable size of nanoparticles also
might realize the EPR effect. IND and PTX had been widely used in
clinical therapy. Therefore, the carrier-free nDDs probably face few
biosafety issues than other nDDs. All of those demonstrated a novel idea
of reverse MDR in NCLS clinical therapy.
Bridging disulfide bond as linker of IND and PTX endowed NPs with the
function of redox-response to target the high-level GSH TME; 2. Nano-
particle was easily obtained and kept stable for at least 20 days. 3. The
size distribution of NPs in aqueous solution was highly uniform with the
narrow value of PDI, and greatly improved the solubility of PTX; 4. IND-
S-S-PTX NPs could obviously reverse MDR by down regulating the
expression of MRP1 protein; 5. IND-S-S-PTX NPs showed obvious cyto-
toxicity and ability to induce apoptosis towards A549/taxol, in addition
to up regulating the generation of intracellular ROS. Furthermore, mo-
lecular simulation was employed to reveal the mechanisms of self-
assembly of IND-S-S-PTX NPs. The results of classic DFT calculations,
IGM analysis and ESP analysis revealed that the driving force of self-
assembly process was mainly originated from van der Waals force.
RDG and a series of molecular dynamics simulation analysis indicated
the spontaneity of the self-assembly process.
CRediT authorship contribution statement
Wenbo Kang: Conceptualization, Methodology, Data curation,
Writing - original draft. Yuanhui Ji: Supervision, Project administra-
tion, Writing - review & editing, Funding acquisition. Yu Cheng: Data
curation.
5. Conclusions
Through the above work, a carrier-free nDDs strategy for NSCLC
therapy was demonstrated. This carrier-free nDDs only consisted of IND-
S-S-PTX NPs aqueous solution which was prepared by nanoprecipitation
method. IND-S-S-PTX NPs showed several unique characteristics: 1.
Declaration of Competing Interest
The authors declare that they have no known competing financial
11

W. Kang et al.
International Journal of Pharmaceutics 603 (2021) 120691
Jain, A., Globisch, C., Verma, S., Peter, C., 2019. Coarse-Grained Simulations of Peptide
Nanoparticle Formation: Role of Local Structure and Nonbonded Interactions.
J. Chem. Theory Comput. 15, 1453–1462.
interests or personal relationships that could have appeared to influence
the work reported in this paper.
Jin, Z., Nguyen, K.T., Go, G., Kang, B., Min, H.K., Kim, S.J., Kim, Y., Li, H., Kim, C.S.,
Lee, S., Park, S., Kim, K.P., Huh, K.M., Song, J., Park, J.O., Choi, E., 2019.
Multifunctional Nanorobot System for Active Therapeutic Delivery and Synergistic
Chemo-photothermal Therapy. Nano Lett. 19, 8550–8564.
Acknowledgements
The authors gratefully acknowledge Dr. Defang Ouyang from Uni-
versity of Macau for the help with quantum chemistry calculations by
using the Gaussian 16 program package and the financial support for
this research from the National Natural Science Foundation of China
(Grant No.: 21978047, 21776046), the Fundamental Research Funds for
the Central Universities (Grant No.: 2242020K40033), and the Six
Talent Peaks Project in Jiangsu Province (Grant No. XCL-079).
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Appendix A. Supplementary material
Supplementary data to this article can be found online at https://doi.
org/10.1016/j.ijpharm.2021.120691.
Martinez-Colon, G.J., Moore, B.B., 2018. Prostaglandin E2 as a Regulator of Immunity to
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