Nanoparticulate delivery of potent microtubule inhibitor for metastatic melanoma treatment

Article abstract of DOI:10.1016/j.jconrel.2019.07.025

Melanoma is the most aggressive type of skin cancer, which readily metastasizes through lymph nodes to the lungs, liver, and brain. Since the repeated administration of most chemotherapeutic drugs develops chemoresistance and severe systemic toxicities, h

Full text of DOI:10.1016/j.jconrel.2019.07.025

Accepted Manuscript
Nanoparticulate delivery of potent microtubule inhibitor for
metastatic melanoma treatment
Jitender Bariwal, Virender Kumar, Hao Chen, Rajan Bhattarai,
Peng Yang, Wei Li, Ram I. Mahato
PII:
S0168-3659(19)30412-2
DOI:
https://doi.org/10.1016/j.jconrel.2019.07.025
COREL 9872
Reference:
To appear in:
Journal of Controlled Release
Received date:
Revised date:
Accepted date:
18 March 2019
9 July 2019
17 July 2019
Please cite this article as: J. Bariwal, V. Kumar, H. Chen, et al., Nanoparticulate delivery
of potent microtubule inhibitor for metastatic melanoma treatment, Journal of Controlled
Release, https://doi.org/10.1016/j.jconrel.2019.07.025
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ACCEPTED MANUSCRIPT
Nanoparticulate Delivery of Potent Microtubule Inhibitor for Metastatic Melanoma
Treatment
1
1
2
1
2*
Jitender Bariwal , Virender Kumar , Hao Chen , Rajan Bhattarai , Peng Yang, Wei Li
1*
and Ram I Mahato
1
Department of Pharmaceutical Sciences, University of Nebraska Medical Center,
Omaha, NE 68198, USA
2
Department of Pharmaceutical Sciences, University of Tennessee Health Science
Center, Memphis, TN 38163, USA
*Corresponding Authors
Ram I. Mahato, Ph.D.
Department of Pharmaceutical Sciences
University of Nebraska Medical Center
9
86025 Nebraska Medical Center
Omaha, NE 68198- 6025
Tel: 402-559-5422; Fax: 402-559-9543
E-mail: ram.mahato@unmc.edu
Wei Li, Ph.D.
Department of Pharmaceutical Sciences,
University of Tennessee Health Science Center
Memphis, TN 38163
Tel: (901)448-7532; Fax: 901-448-6828
Email: wli@uthsc.edu

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ABSTRACT
Melanoma is the most aggressive type of skin cancer, which readily metastasizes
through lymph nodes to the lungs, liver, and brain. Since the repeated administration of
most chemotherapeutic drugs develops chemoresistance and severe systemic
toxicities, herein we synthesized 2-(4-hydroxy-1H-indol-3-yl)-1H-imidazol-4-yl)(3,4,5-
trimethoxyphenyl) methanone (abbreviated as QW-296), a novel tubulin destabilizing
agent with little susceptible to transporter-mediated drug resistance. QW-296 disturbed
the microtubule dynamics at the nanomolar concentration in A375 and B16F10
melanoma cells. QW-296 binding to colchicine-binding site on tubulin protein was
confirmed by molecular modeling and tubulin polymerization assay. QW-296
significantly inhibited A375 and B16F10 cell proliferation, induced G2/M cell cycle arrest
and led to apoptosis and cell death. To improve its aqueous solubility, QW-296 was
encapsulated into methoxy poly(ethyleneglycol)-b-poly(carbonate-co-lactide) [mPEG-b-
P(CB-co-LA)] polymeric nanoparticles by solvent evaporation, with the mean particle
size of 122.0±2.28 nm and drug loading of 3.70% (w/w). Systemic administration of
QW-296 loaded nanoparticles into C57/BL6 albino mice bearing lung metastatic
melanoma at the dose of 20 mg/kg 4 times a week for 1.5 weeks resulted in significant
tumor regression and prolonged mouse median survival without significant change in
mouse body weight. In conclusion, QW-296 loaded nanoparticles have the potential to
treat metastatic melanoma.
Keywords:
Microtubule/tubulin
inhibitor,
melanoma,
nanoparticles,
tubulin
polymerization

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INTRODUCTION
Microtubules are an attractive target for chemotherapy due to their critical role in
1,2
mitosis, cellular shape maintenance, and intracellular transportation. Microtubule has
the following three distinct binding domains: vinca binding domain, taxane binding
2
domain, and colchicine binding domain. Due to the dynamic nature of tubulin,
chemotherapeutic agents that promote microtubule polymerization are classified as
microtubule stabilizers, whereas agents that inhibit microtubule polymerization are
designated as tubulin destabilizing agents. Chemotherapeutic agents that bind to
taxane binding site are microtubule stabilizers, whereas agents that bind to vinca or
3
colchicine binding site are tubulin destabilizers. Clinical potential of tubulin binding
agents has been proven by many Food and Drug Administration (FDA) approved
anticancer drugs including paclitaxel (PTX), docetaxel (DTX), and epothilones that bind
to taxane binding domain, whereas vinblastine, vincristine, and vinorelbine bind to vinca
binding domain. Combretastatin (CA) and colchicine bind to the colchicine binding
4-7
domain.
Clinical efficiency of drugs that bind to vinca or taxane binding domain is
limited due to the development of chemoresistance by overexpressing transmembrane
efflux pumps {P-glycoprotein (P-gp), multidrug resistance-associated protein (MRP)
8-10
and/or breast cancer resistance protein (BCRP)} in cancer cells.
Interestingly, the
compounds interacting with the colchicine binding site has been reported to be less
11
sensitive to clinically observed mechanisms of resistance. Despite the clinical use of
colchicine for the treatment of gout, its use as an anticancer drug is limited due to toxic
12
side effects.

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Among various malignant cancers, melanoma has a very high metastatic
tendency in the late stages where cancer cells metastasize to the lymph nodes, lungs,
liver, brain, and heart. This metastatic property of melanoma leads to the low survival
13
time of patients typically less than a year. Dacarbazine (DTIC) is a first-line FDA-
approved DNA alkylating agent used for treating advanced metastatic melanoma.
14
However, it has not significantly improved the patient overall survival time , and only 1–
15
2
% of patients respond well. The effectiveness of PTX and carboplatin combination, a
second-line FDA-approved therapy, has only less than 40% of all the patients
16,17
responded well while receiving this combination.
Treatment of metastatic melanoma
with the FDA approved BRAF inhibitors such as vemurafenib or dabrafenib with
advanced BRAFV600 mutated melanoma has shown high response rate initially,
however the effect last for a limited period. Similar results were observed when treated
18
with MEK inhibitors such as trametinib. Further, the treatment of metastatic melanoma
with immunomodulating drugs such as anti-PD-1 drugs (nivolumab, pembrolizumab) or
anti-CTLA-4 antibody ipilimumab resulted in improved progression-free survival.
However, treatment with these drugs results in the adverse effect of grade 3-4, including
severe elevation in liver injury markers, life-threatening skin rashes, nephritis, and
19
pneumonitis.
Clinical efficiency of chemotherapeutic drugs is limited by off-target toxicity and
20,21
development of chemoresistance by multiple mechanisms.
metastasizing cancers like melanoma may be dealt by addressing the following issues:
1) discovery of potent molecules acting on vital cell proliferation mechanism and block
the cell migration/invasion (2) overcome the cellular resistance mechanism and (3)
Aggressively
(

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reduced off-target toxicity at the therapeutic dose. In this context, limited available
therapeutic options for the treatment of metastatic melanoma, the high therapeutic
efficiency of tubulin inhibitors, and relatively low sensitivity to clinical resistance,
colchicine binding site offers a potential target to inhibit the tubulin protein in rapidly
dividing and metastasizing cells.
Our group is continuously involved in the discovery and development of new
small molecules to target the colchicine binding site of tubulin. Previously, we reported
22
the discovery of diaryl-ketone chemotypes, with various linkers such as a phenyl ring,
23
2
4
-substituted
methoxybenozyl
arylthiazoles,
arylbenzoylimidazoles,
phenylaminothiazoles,
and
24
25
arylbenzoylimidazoles,
reverse
1H-imidazo[4,5-
3
c]pyridine, that selectively binds to the tubulin at colchicine binding site and stabilizes
the tubulin at nanomolar concentration.
Nanoparticulate delivery systems have the potential to deliver drugs to the target
26
site and prevent off-target toxicity by a passive delivery mechanism utilizing the
27
Enhanced Permeability and Retention (EPR) effect. Beside this, nanoparticles also
improve pharmacokinetic profiles by improving the aqueous solubility of the hydrophobic
drug, extending circulation time and improving bioavailability. We successfully applied
28
micellar delivery for 4-substituted methoxybenzoyl-aryl-thiazole-100 (SMART-100) and
29
bicalutamide to improve their aqueous solubility using methoxy poly(ethylene glycol)-
b-poly(D, L-lactide) (mPEG-PLA) polymer. Despite improved solubility, low drug loading
in micelles prepared from mPEG-PLA based polymer shifted our interest to develop
new, safe and biocompatible polymers. We utilized methoxy poly(ethyleneglycol)-b-
poly(carbonate-co-lactide) [mPEG-b-P(CB-co-LA)] based nanoparticles to deliver (2-(1

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H-indol-5-yl) thiazol-4-yl) 3, 4, 5-trimethoxyphenyl methanone (LY293) to treat
melanoma. LY293-loaded nanoparticles significantly inhibited the proliferation of
metastasized melanoma in a lung metastatic melanoma mouse model without toxicity to
30,31
vital organs.
In our recent studies, we synthesized a new SMART analog, SMART-OH
(
Figure 1A) and conjugated with methoxy poly(ethylene glycol)-b-poly(2-methyl-2-
carboxyl-propylene carbonate-g-dodecanol) (mPEG-b-PCC-g-DC) to improve the drug
payload and prevent premature drug release. The polymeric micelles with 14.3% drug
payload, showed significant inhibition of tumor growth in the lung metastatic melanoma
32
mouse model. In another study, we conjugated amino derivative of indocyanine green
ICG-NH2) with mPEG-b-PCC-g-DC for photodynamic and photothermal effects on
melanoma cells. ICG conjugated micelles exhibited excellent in vivo stability and
(
33
significant improvement in the therapeutic efficacy of ICG mediated phototherapy.
Herein, we synthesized a new potent tubulin inhibitor 2-(4-hydroxy-1H-indol-3-yl)-
H-imidazol-4-yl)(3,4,5-trimethoxyphenyl)methanone (abbreviated QW-296 3) bearing a
-hydroxyl group on the indole ring of ABI-III 2 (Figure 1A). Because of the low
1
4
resistance index of QW-296 in taxane-resistant prostate cancer cells, compared to ABI-
III, we pursued QW-296 for further in vivo efficacy. QW-296 loaded nanoparticles were
prepared using our previously optimized {mPEG-b-P(CB-co-LA)} polymer and
demonstrated its promising therapeutic efficiency in lung metastatic melanoma bearing
C57/BL6 albino mice.
Materials and Methods
Materials

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Dulbecco’s Modified Eagle’s Medium (DMEM) was purchased from Invitrogen
(
Carlsbad, CA). All primary antibodies were purchased from Cell Signaling (Beverly,
MA) and Santa Cruz Biotechnology (Dallas, TX). B16F10 mouse melanoma cell line
and A375 human melanoma cell line were acquired from the American Type Culture
Collection (ATCC). Cells were cultured in DMEM supplemented with 10% fetal bovine
serum (FBS) (Atlanta Biologicals), and 1.0% antibiotic/antimycotic mixture (Invitrogen)
PC3 and PC3-TXR cells were kindly provided by Dr. Evan T. Keller from the University
of Michigan. These cells were cultured in RPMI-1640 medium supplemented with 10%
FBS and 1% penicillin/streptomycin in a humidified incubator containing 5% CO2 at 37
°
C and their resistance to taxane was maintained by adding 200 nM paclitaxel to growth
media biweekly. 2,2-Bis(hydroxymethyl)propionic acid, methoxy poly(ethylene glycol)
mPEG, 5,000 Da), 8-diazabicycloundec-7-ene (DBU), benzyl bromide, L-lactide, and all
(
other reagents were purchased from Sigma-Aldrich (St. Louis, MO) and used as
received.
Synthesis of QW-296
34
Synthesis of QW-296 (Figure 1A) was achieved as described previously.
Briefly, QW-296 was synthesized from compound 1 that reacts with POCl3 and DMF to
give compound 2 followed by reaction with benzene sulfonyl chloride to afford
compound 3 as a major product. Compound 3 will react with ammonium hydroxide and
glyoxal to form compound 4. Subsequent compound 4 reacts with NBS in THF provided
the dibromo imidazole derivative 5. Protection of the imidazole in the presence of
SEMCl and NaH afforded intermediate 6 which could be smoothly reacted with 3,4,5-
Trimethoxybenzoyl chloride and isopropylmagnesium chloride-lithium chloride complex

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to give 7, and then removal of benzenesulfonyl and bromo group provided the
intermediate 8. Subsequent deprotection of the SEM group with 1 M HCl provided the
compound 9, which after removal of benzyl group provided the target compound QW-
2
96 as a yellow solid. The chemical structures of the synthesized compounds were
characterized and confirmed by NMR and high-resolution mass spectrometry. The
1
13
characterization data of QW-296 by H NMR, CNMR, HRMS, and HPLC are shown in
supplementary Figures 3S and 4S.
Docking studies
Molecular Modeling. The molecular modeling studies were performed with the
published crystal structure of DJ101 in complex with tubulin at a resolution of 2.8Å (PDB
code: 5H7O). Schrödinger Molecular Modeling Suite 2018 (Schrödinger LLC, New York,
3,35
NY) was used for the modeling studies with similar procedures described before.
Briefly, the structures of the protein-ligand complexes were prepared using the Protein
Preparation Wizard workflow. The tubulin receptor grid was generated based on the
native ligand using the Receptor Grid Generation. Both ABI-III and QW-296 tubulin
inhibitors were built and prepared for docking using the Ligprep module before they
were docked into 5H7O. The Glide docking score was used to show the estimated free
energy of binding (kcal/mol); a lower (more negative) number indicates more favorable
interaction. Hydrogen bonds and data analysis were carried out using the Maestro
interface of Schrödinger software.
Tubulin Polymerization Assay

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In vitro tubulin polymerization assay was performed using the fluorescence-
based tubulin polymerization kit purchased from Cytoskeleton, Inc. (Denver, CO). The
polymerization assay is a one-step assay, which uses a half area 96-well plate (Corning
Costar, Corning, NY) for measuring tubulin polymerization. A 5 L aliquot of a 10X
concentration of the test compound in DI was pipetted into each well of the pre-warmed
9
6-well plates at 37 °C. Following the addition of compound, 50 L of tubulin MIX was
added (as per manufacturer’s protocol). Then, the plate was immediately subjected to
e
kinetic measurements on a SpectraMAx M5 fluorescence microplate reader (Molecular
Device, San Jose, CA). The kinetics of tubulin polymerization reaction was monitored
for every 30 s over a period of 90 min at 37 °C with the medium shaking of the plate for
5
s before taking the first reading. The change in the intensity of fluorescence due to
polymerization or depolymerization of tubulin was determined by setting the excitation
wavelength at 360 nm and emission wavelength at 450 nm using medium gain.
Reaction without any compound (DMSO, 2%) and tubulin protein served as negative
controls. All experiments were performed in triplicates.
P-gp ATPase Activity Assay
Pgp-Glo assay system (Promega, Madison, WI) was used to evaluate the effect
of QW-296 on Pgp ATPase activity according to the manufacturer’s protocol. In a 96-
well untreated white plate, QW-296 was incubated at the dose of 10, 100 and 1000 nM
with 25 μg of recombinant human Pgp membrane. Pgp-GIo assay buffer was used as
the untreated control, 200 μM verapamil was used as the positive control of drug-
induced Pgp ATPase activity, and 100 μM sodium orthovanadate was used as the
selective inhibitor of Pgp ATPase activity. ATPase activity was initiated by the addition

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of 5 mM Mg ATP and incubated at 37 °C for 40 min. Further, luminescence was initiated
by adding 50 μL of ATP detection reagent; after 20 min of incubation at room
e
temperature, the plate was read on SpectraMAx M5 microplate reader (Molecular
Device, San Jose, CA).
Synthesis of mPEG-b-P(CB-co-LA)
28
Benzyl-2,2-bis(methylol)propionate was synthesized as reported previously by
reacting 2,2-bis(hydroxymethyl)propionic acid 4 with benzyl bromide 5 at 100 °C for 15
h. The monomer 5-methyl-5-benzyloxycarbonyl-1,3-dioxane-2-one (MBC) 8 was
synthesized by reacting benzyl-2,2-bis(methylol)propionate 6 with triphosgene 7 at −70
°C in the presence of pyridine in dry dichloromethane. Random MBC and lactide block
copolymer mPEG-b-P(CB-co-LA) 11 with target average molecular weight of 15,000 Da
was synthesized by the ring-opening polymerization of L-lactide 10 and MBC 8 using
mPEG 9 as a macroinitiator and DBU (2.5 mol% relative to mPEG) as a catalyst in dry
DCM at room temperature for 3.5 h. Following synthesis, the copolymer was
1
characterized by H NMR and gel permeation chromatography (GPC). The molecular
weight and polydispersity index (PDI) of mPEG-b-P(CB-co-LA) polymers were
determined by GPC using a Shimadzu gel permeation chromatography (GPC) system
equipped with a Styragel HR 4E GPC column and a differential refractive index
detector. Dimethylformamide (DMF) with 0.1% LiBr was used as eluent at a flow rate of
0
.6 mL/min at 40 °C. Narrow PEG standards (3,770- 71,000 g/mol) from American
Polymer Standards Corp. were used for generating a standard curve, and the data was
processed using Lab Solutions software ver. 5.84.
Preparation and characterization of nanoparticles

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Blank and QW-296 loaded nanoparticles were prepared by oil/water (o/w)
emulsification using 1.0% polyvinyl alcohol as an emulsifier. mPEG-b-P(CB-co-LA) and
QW-296 (an amount equivalent to 10% theoretical drug loading) were dissolved in a
solvent mixture of dichloromethane and acetone (1:1) and were emulsified using a
sonica probe sonicator (Newtown, CT) at an amplitude of 20 W for 10 min. The organic
phase was removed using a rotary evaporator, followed by hydration and centrifugation
at 5000 rpm for 5.0 min. The supernatant was concentrated by ultracentrifugation at
2
0,000 rpm for 30 min followed by washing with PBS. Then, the sediment was
suspended in the desired volume of PBS. The particle size distribution and ζ potential of
drug-loaded nanoparticles were determined by dynamic light scattering (DLS) using a
Malvern Zetasizer (Worcestershire, UK). Surface morphology and particle size of
nanoparticles were determined by Atomic Force Microscopy (AFM). Briefly, nanoparticle
samples were diluted in PBS, deposited on freshly cleaved mica modified with 1-(3-
aminopropyl)-silatrane (APS), rinsed with de-ionized (DI) water, and dried with a gentle
flow of argon. Images were collected with MultiMode Nanoscope IV system (Bruker
Instruments, Santa Barbara, CA) in Tapping Mode at ambient conditions. Silicon probes
RTESPA-300 (Bruker Nano Inc., CA) with a resonance frequency of ~300 kHz and a
spring constant of ~40 N/m were used for imaging at scanning rate for about 2.0 Hz.
Images were processed using the FemtoScan software package (Advanced
Technologies Center, Moscow, Russia).
Drug Loading and Encapsulation
The encapsulation efficiency was estimated by reverse-phase high-performance
liquid chromatography (RP-HPLC) at λmax of 222 nm using a C18 column (250 mm X 4.6

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mm, 5µ, Alltech, Deerfield, IL), a solvent mixture of acetonitrile and water (55:45 v/v)
was used as a mobile phase. QW-296 concentration was calculated using the peak
2
area from a standard curve with R = 0.9992 and a quantification limit of 1.95 g/mL.
Drug content was determined by taking 500 µl of nanoparticles in 1 ml of
dichloromethane (DCM) and sonicated in a bath sonicator for 15 min at room
temperature to extract the drug content in DCM. Then, 500 µl DCM was taken in a glass
vial, and DCM was removed under reduced pressure on a rotary evaporator and
reconstituted in 1.0 ml of acetonitrile. The acetonitrile solution of QW-296 was filtered
through a 0.2 µm syringe filter (Acrodisc, Waters, USA) and 20 µl of the filtered solution
was injected into the HPLC system.
In vitro release of QW-296 from nanoparticles
QW-296 release from mPEG-b-P(CB-co-LA) nanoparticles was determined by
placing 3.0 ml of formulated nanoparticles in a dialysis bag (1,000 Da cutoff) and
dialyzed against 50 mL PBS (pH 7.4) containing 1.0% Tween 80 as a nonionic
surfactant and emulsifier at 37°C in a temperature controlled shaker at the speed of 100
rpm. Sample (1.0 mL) was taken between 0 - 97 h and replaced with 1.0 mL PBS
containing 1.0% Tween 80. The sample was dissolved in the mobile phase after
removing the solvent using an evaporator, followed by determination of drug
concentration using HPLC (mobile phase: Acetonitrile:water (55:45), C18 column (250 ×
4
.6 mm, 5µ, Alltech, Deerfield, IL), flow rate 1 mL/min). All experiments were performed
in triplicate at different days, and the data reported as the mean ± SEM of three
individual experiments.
Cell viability assay

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A375 and B16F10 cells were seeded overnight in 96-well plates at a density of
,500 cells per well and thereafter treated with DMSO, QW-296 dissolved in DMSO,
2
and SMART-OH dissolved in DMSO for 48 h. At the end of 48 h, the cell culture
medium was replaced with 100 μL of 0.5 mg/mL 3-(4,5-dimethyl-thiazol-2-yl)-2,5-
diphenyl tetrazolium bromide (MTT) solution in PBS and incubated for 4 h at 37 °C. Cell
culture media was removed, and formazan crystals were dissolved in 200 μL of DMSO.
The absorbance was measured at 560 nm. Similarly, PC3 and PC3-TXR cells were
treated with PTX, QW-296, and ABI-III to calculate the IC50 values.
Cell cycle analysis
6
A375 and B16F10 cells were cultured in a six-well plate at a density of 0.2×10
cells per well. After reaching the confluency to 80%, the cells were treated with DMSO,
QW-296 cosolvent and QW-296 loaded nanoparticles for 48 h. Cells were harvested,
fixed in 70% ice-cold ethanol for 1 h and washed with PBS. A cell pellet containing
6
1
×10 cells was suspended in 0.5 ml of FxCycle™ PI/RNase staining solution for 15 min
at room temperature. Cell cycle was measured by a flow cytometer (BD FACS Calibur,
NJ). Each experiment was carried in triplicate at different days and the data reported as
the mean ± SEM.
Apoptosis Signaling by Annexin-V
A375 and B16F10 cells were seeded in 6 well plates overnight at the density of
6
0
.2×10 cells per well and treated with DMSO, QW-296 cosolvent and QW-296 loaded
nanoparticle for 48 h. Then, cells were washed twice with cold PBS, and the pellets
were suspended in 500ꢀµL binding buffer containing 5.0ꢀµL of Annexin V-FITC and 5ꢀµL
of propidium iodide (Annexin V-FITC Apoptosis Kit, BioVision Inc., Milpitas, CA). The

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mixture was incubated for 5ꢀmin in the dark at room temperature and analyzed by flow
cytometry (Ex = 488nm; Em = 530 nm). Each experiment was performed in triplicate at
different days, and the data reported as the mean ± SEM.
Western blot analysis
A375 and B16F10 cells were cultured inoculated in a 6-well plate at the density
6
of 0.2×10 cells per well and after 80% confluency, cells were treated with DMSO, QW-
2
96 cosolvent, colchicine and QW-296 loaded nanoparticle for 48 h. Then, cells were
washed twice with cold PBS, lysed with RIPA buffer, and protein concentrations were
measured with bicinchoninic acid (BCA) protein assay kit (Pierce, Rockford, IL). The
lysate was boiled for 5 min, subjected to a 15 % SDS-PAGE, and transferred to a PVDF
membrane using the iBlot™system (Invitrogen, Carlsbad, CA). Membranes were
blocked with 3.0 % BSA in 1X Tris-buffered saline (TBS) at room temperature for 1.0 h
and then incubated with primary antibodies at 4 °C overnight, followed by incubation
with anti-mouse, anti-goat or anti-rabbit IRDye 800CW secondary antibodies for 1.0 h at
RT in dark conditions. All the blots were probed with total β-actin or GAPDH antibody as
a control. The signal of target proteins was detected Li-COR Odyssey® infrared imaging
system (Li-COR, Lincoln, NE).
Confocal Microscopy/ Immunofluorescence
A375 and B16F10 cells were seeded on eight culturewell-chambered cover glass
coverslips at a density of 2000 cells/well and were incubated overnight. After treatment
of cells with DMSO, QW-296, and colchicine, for 24h, cells were washed twice with ice-
cold PBS. Cells were fixed with 4% formaldehyde solution overnight at 4 °C. Then, cells

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were washed three times with PBS to remove the formaldehyde completely. Cells were
then incubated in the mixture (1:1) of 2.0% BSA (in PBS) and 0.1% Triton X-100 (in
PBS) for 1.0 h at room temperature. Then, cells were washed three times with PBS and
incubated the cells with anti--tubulin antibody (Thermo Scientific, Rockford, IL) at 4
°C overnight. Cells were then washed three times with PBS to remove the primary
antibody completely. After this, cells were incubated with secondary antibody (Goat anti-
Rabbit IgG-FITC, Santa Cruz Biotechnology, Dallas, TX) for 1.0 h at room temperature.
Then, cells were washed three times with PBS to remove the secondary antibody
completely. Cells were then stained with a solution of DAPI (1.0 l of DAPI in 200 l of
0
.1% Triton X-100) and incubated for 10 min at room temperature. Finally, cells were
washed three times with PBS and images were acquired with a Zeiss 710 confocal
microscope and Zen imaging software (Zeiss, Thornwood, NY).
Scratch migration assay
5
A375 and B16F10 cells were seeded in 24-well plates (2×10 cells/well) in
replicates of three and incubated overnight. Using a 200 µL pipette tip, a straight line
was scratched through the cell monolayer to remove an area of cells, and cells were
washed several times to remove any debris and uprooted cells. Media was replaced
with media containing vehicle (DMSO), and 60 nM of colchicine or QW-296. Images
were obtained after 0, and 24 hours with an inverted microscope at 10X magnification.
Terminal Deoxynucleotidyl Transferase Nick End Labeling (TUNEL) Staining
Terminal
deoxynucleotidyl
transferase
(TdT)
mediated-16-deoxyuridine
triphosphate (dUTP) Nick-End Labelling (TUNEL) system (Promega, USA) was used to

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detect apoptosis in lung tumor tissue sections. Briefly, paraffin-embedded tumor tissue
sections were deparaffinized by immersing in xylene and rehydrated by sequentially
immersing the slides through graded ethanol. After washing with PBS, sections were
fixed with 4% formaldehyde, washed with PBS, and permeabilized by incubating with
Proteinase K solution (20 µg/mL) for 15 min. Afterward, slides were covered with 100 μL
of equilibration buffer for 15 min and washed with PBS. Slides were coated with rTdT
incubation buffer (equilibration buffer + nucleotide mix + rTdT enzyme) and incubated at
3
7 °C for 60 min. Endogenous peroxidases were blocked using 0.3% hydrogen
peroxide. For the positive control, the sample was treated with DNase I (5-10 units/ml)
in DNase I buffer for 15 min. Tissue sections were incubated with streptavidin-
conjugated horseradish peroxide (HRP) at room temperature for 30 min. Then, the
visualization of fragmented DNA was performed by treating the samples with a solution
of hydrogen peroxide and diaminobenzidine (DAB).
In vivo study
All animal experiments were performed following the NIH animal use guidelines
and protocol approved by the Institutional Animal Care and Use Committee (IACUC) at
the University of Nebraska Medical Center (UNMC), Omaha, NE. A mouse model of
metastatic melanoma to the lung was established in 8-10 weeks old female C57BL/6
5
albino mice by injecting 2×10 B16F10-Luc cells suspended in 100 μl PBS in the tail
vein. Mice were randomly divided into three groups of six animals per group when the
6
2
radiance of the tumor had reached 10 p/sec/cm sr. QW-296 in cosolvent and QW-296
loaded nanoparticles were administered intravenously to mice once every three days for
a total of five times. Group 1 was kept as the control and received blank nanoparticles,

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group 2 received 20 mg/kg QW-296 in 35% of cosolvent (50% propylene glycol, 30%
Cremophor® EL, and 20% ethanol) and 65% of dextrose solution, and group 3 received
nanoparticle formulation equivalent to free 20 mg/kg of QW-296. Bioluminescent
radiance of tumor was measured every other day using the IVIS® Spectrum imaging
system (PerkinElmer Inc., MA). Mice were sacrificed, tumors and other major organs
such as liver, spleen, kidney and heart were isolated. Three representative tumor
tissues were collected per group and fixed with 10% buffered formalin for 24 h. The
fixed samples were embedded in paraffin and thin sections of 4.0 μm were obtained and
immunostained for hematoxylin and eosin (H&E), Ki67 and cleaved Caspase 3.
Statistical analysis
Data represented as the mean ± S.D. The statistical comparisons of the
experiments were performed by two-tailed Student's t-test. P<0.01 was considered
statistically significant.
Results
Synthesis of QW-296
QW-296 (Figure 1A) was synthesized using the starting 4-(benzyloxy)-1H-indole
rd
as a starting material, which was formylated at 3 position. Subsequently, after
rd
protecting indole nitrogen, imidazole group was introduced at the 3 position. Imidazole
was then brominated, and the imidazole nitrogen was protected followed by
displacement of the 3-bromo group with the trimethoxybenzoly group. Subsequently, a
remaining bromine atom and both protecting groups were removed to get QW-296 as a
yellow solid. LCMS, NMR, and HRMS confirmed the purity and chemical structures of

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1
QW-296. H NMR (400 MHz, DMSO-d6) δ 14.02 – 13.06 (m, 2H), 11.59 (dd, J = 65.1,
2
7
.9 Hz, 1H), 8.86 – 8.07 (m, 1H), 8.07 – 7.81 (m, 1H), 7.60 – 7.12 (m, 2H), 7.01 (td, J =
.9, 5.2 Hz, 1H), 6.87 (dd, J = 8.1, 4.5 Hz, 1H), 6.41 (dd, J = 13.9, 7.6 Hz, 1H), 3.91 (d,
13
J = 4.9 Hz, 6H), 3.79 (d, J = 4.4 Hz, 3H). C NMR (101 MHz, DMSO-d6) δ 185.08,
1
1
1
1
3
82.08, 152.79, 152.54, 151.76, 151.54, 148.71, 145.69, 141.07, 140.83, 138.57,
38.54, 137.56, 136.29, 133.22, 132.86, 130.25, 126.11, 125.15, 124.29, 124.25,
23.29, 113.93, 113.73, 106.57, 105.96, 105.47, 105.28, 105.04, 104.73, 102.53,
+
02.43, 60.11, 55.97, 55.73, 54.89. HRMS [C21H20N3O5 ]: calcd 394.1403, found
94.1414. HPLC purity 99.1% (tR = 3.03 min). (Supplementary Figure 3S-4S).
Copolymer Synthesis and preparation of QW-296 loaded nanoparticles
We utilized our previously developed mPEG-b-P(CB-co-LA) copolymer
30,31
to
prepare QW-294 loaded nanoparticles (Figure 1B) owing to ease in synthesis,
biocompatibility, and ability to form stable nanoparticles. The degree of polymerization
1
and molecular weight of this copolymer was confirmed by H NMR spectra (Figure 1S).
1
From H NMR, the calculated molecular weight of this copolymer was 13,000 Da with a
low polydispersity index (PDI) of 1.118. The mean particle size of QW-294 loaded
nanoparticles was 122.0 ± 2.28 nm with PDI of 0.153 as determined by DLS (Figure
1
C). Surface morphology and particle size of these nanoparticles were determined by
AFM, and  potential by Malvern Zetasizer. These nanoparticles were of spherical
shape and had narrow particle size distribution, with the mean particle size of 70.02 nm
(Figure D) and  potential of zero mV. These results were included in Figure 1 and
Supplementary Figure 2S. The Drug loading determined by HPLC was 3.70% w/w at

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the theoretical loading of 10.0%. There was no batch to batch variation in particle size
and drug loading.
In vitro release profiles of QW-296 loaded nanoparticles
The release profile of QW-296 from nanoparticles was determined by dialysis
and results are shown in Figure 1E. There was no burst effect and only 7.9% of QW-
2
96 was released at pH 7.4 after 4.0 h of dialysis, but almost 68.3% release was
achieved after 97.0 h suggesting sustained drug release over a long period.
A
B
D
ii
i
1
5
0
5
0
1
0
20 40 60 80 100 120 140 160 180
Diameter (nm)
E
8
6
4
2
0
0
0
0
0
C
0
50
Time (h)
100
Size (d. nm)
Figure 1. Structure of colchicine inhibitors, mPEG-based polymer synthesis, and
nanoparticle characterization. A) Chemical structure of SMART-OH, ABI-III and QW-
2
96, B) Synthesis of mPEG-poly(carbonate-co-lactide). C) The particle size distribution
of polymeric nanoparticles by w/o emulsification as determined by dynamic light
scattering (DLS). D) Surface morphology and particle size distribution of nanoparticles
by AFM. E) In vitro release of QW-296 from nanoparticles at pH 7.4. Drug
concentrations were measured by HPLC. Results are expressed as the mean ± SD (n =
3
).

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Molecular modeling and effect on tubule polymerization
To confirm the binding interaction of QW-296 with colchicine binding site, we
used molecular modeling studies using high-resolution crystal structure of DJ101 in
complex with tubulin. Molecular modeling confirmed that QW-296 has similar binding
interactions as the native compound ABI-III, and binds to the same binding site in
tubulin protein (Figure 2A).
To confirm the binding site of QW-296 on tubulin, we conducted in vitro tubulin
polymerization assay. Tubulin (3 mg/ml) was exposed to 5 μM QW-296, colchicine,
SMART-100 or vehicle control (2% DMSO), respectively, and incubated in tubulin
buffer. Both QW-296 and colchicine effectively inhibited polymerization, while robust
polymerization was observed in SMART-OH-treated group (Figure 2B). These results
indicated that QW-296 is a strong tubulin depolymerizing agent. We also determined the
effect of QW-296 on microtubule networks by confocal microscopy of A375 and B16F10
cells treated for 18 h with QW-296, using colchicine as a strong inhibitor of tubulin
polymerization as a positive control. There was a significant alteration in microtubule
arrangement in the treated cells (Figure 2C). Control cells exhibited well-dispersed
microtubule networks throughout the cytoplasm, whereas the cells treated with QW-296
had scattered microtubule networks with weaker fluorescent signal due to an increase in
dispersed cytoplasmic fluorescence, like colchicine-treated cells. These observations
further confirm that QW-296 exhibits strong tubulin binding affinity at colchicine binding
site.

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A
B
DMSO
Colchicin 5 µM
QW-296, 5 µM
SMART-OH 5 µM
1
000
α-Thr179
ABI-III
ɑ-subunit
8
6
4
2
00
00
00
00
0
β-Lys350
β-subunit β-Asp249
QW-296
β-Cys239
Time (s)
Colchicine (nM)
60
QW-296 (nM)
C
DMSO
4
0
60
4
0
Figure 2. Confirmation of the mechanism of action of QW-296. A) Proposed binding
poses of ABI-III and QW-296 in the tubulin crystal structure (PDB code: 5H7O).
Superposition of ABI-III (purple tube model; glide docking score -11.2) with QW-296
(orange tube model; glide docking score -11.9). B) Effect of QW-296 on tubulin
polymerization in in vitro. C) Confocal images of A375 and B16F10 melanoma cells
exposed to colchicine and QW-296 or the vehicle (DMSO) for 24 h.
Anticancer activity
Cytotoxicity of QW-296 was determined in A375 and B16F10 cells by incubating
cells at different drug concentrations. As shown in Figure 3A and 3B, there was dose-
dependent cell killing, with the IC50 values of 30 and 35 nM, respectively. Since QW-296
was more effective in inhibiting tubulin than SMART-OH, we decided to compare QW-

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2
96 and SMART-OH for their dose-dependent cytotoxic effect on A375 cells. As shown
in Figure 3C, QW-296 was significantly more effective in cell killing resulting in IC50
value of 30 nM compared to that of 350 nM for SMART-OH. Therefore, we decided to
focus on QW-296 for all subsequent studies.
A
B
1
1
20
00
120
100
80
60
40
20
0
QW-296 Free Drug treatment to A375 cells
QW-296 Free Drug treatment to B16F10
8
6
4
2
0
0
0
0
0
0
20
40
60
80
100
120
140
0
20
40
60
80
100
120
140
Conc. (nM)
Conc. (nM)
C
1
1
20
00
QW-296
SMART-OH
80
60
40
20
0
0
50 100 150 200 250 300 350 400 450 500
Conc. (nM)
Figure 3. Cell viability assay using QW-296 in melanoma cells. A) Cytotoxicity of QW-
96 in A375 cells, B) in B16F10 cells and C) comparison of cytotoxicity of QW-296 and
2
SMART-OH in A375 cells after 48 h of treatment.
Cell cycle arrest and apoptosis
Cell cycle arrest was determined by propidium iodide (PI) staining after 24 h of
treatment with QW-296 in cosolvent and nanoparticle formulations in A-375 and B16F10
cells. For A375 cells, results showed that treatment with free QW-296 at 40 and 60 nM
caused 74.4 and 88.7% cell arrest in G2 phase after 24 h of treatment, whereas small
number of cells were arrested in S-phase (21.5% and 9.0%, respectively) and G1 phase
(4.1 and 2.3%, respectively). Treatment of these cells with QW-296 loaded

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nanoparticles (40 and 60 nM equivalent concentration) resulted in enhanced cell arrest
in G2 phase 90.9% vs 74.4 % at 40 nM and 92.1% vs 88.7% at 60 nM. Further, the
number of cells in S-phase (7.8% and 6.9%, respectively) and G1 phase (1.3% and
1
.0%, respectively) were reduced. (Figure 4A)
Treatment of B16F10 cells with free QW-296 at 40 and 60 nM for 24h caused 74
and 76.3% cell arrest in G2 phase, whereas only 14.9.5% and 16.8% of these cells
were arrested in S-phase, and 11.1 and 6.9%, respectively in G1 phase (Figure 4B).
Treatment of these cells with QW-296 loaded nanoparticles at 40 and 60 nM equivalent
doses resulted in an equal number of cell arrest in G1 phase (74.5% vs. 74 % at 40 nM
concentration and whereas at 60 nM concentration increased the number of cells were
arrested in the G2 phase (81.4% vs. 76.3%). Further, similar numbers of cells were
observed in S-phase (15.8% and 13.7% respectively) and G1 phase (7.1% and 4.8%
respectively) (Figure 4B).
To determine whether QW-296 binds to tubulin and arrests the cell cycle in G2/M
35
phase, A375 and B16F10 cells were treated with QW-296 and probed for cyclin B and
36
PTTG.
Treatment with QW-296 and colchicine upregulated PTTG and cyclin B1
expression compared to non-treated cells (Figure 4C), suggesting QW-296 could block
37
the cell cycle at the G2/M phase.
Total apoptotic cell populations significantly increased from 0.1% in the non-
treated control to 13.55% and 20.4% when A375 cells were incubated with QW-296
formulated in cosolvent at 40 and 60 nM, respectively. A similar number of cells were
found apoptotic when treated with an equivalent concentration of QW-296 loaded
nanoparticles (Figure 4D). For B16F10 cell, apoptotic cell population was significantly

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increased when the cells were treated with QW-296 formulated in cosolvent at 40 and
6
0 nM compared to the control group (4.95% vs. 14.55 and 15.5%, respectively),
Further, the apoptotic cell population increased to 16.7% and 20%, respectively when
the cells were treated with QW-296 loaded nanoparticles (Figure 4E). However, there
was statistically no difference between cosolvent and nanoparticle formulation treated
groups.
A 120
G1%
G2%
S%
D
1
00
*
** ** **
ns
*
*
** **
25
20
80
60
40
20
0
**
#
†
*
#
1
5
*
*
*
** ** **
10
5
DMSO NP
Ctr
FD
40 nM
G1% G2% S%
NP
FD
NP
60 nM
1
1
20
00
0
B
ns
*
*
**
**
ctr
40 nM
FD NP
60 nM
*
*
*
*
*
**
80
60
40
20
0
E 2
5
0
ns
#
†
2
#
*
*
FD
*
*
*
*
**
FD
15
*
*
NP
DMSO NP
Ctr
NP
1
0
5
0
40 nM
60 nM
QW-296 (nM)
40
60
C
Colchicine (nM)
DMSO 40
60
PTTG
Ctr
40 nM
60 nM
Cyclin B1
GAPDH
FD
NP
Figure 4. Effect of QW-296 on cell cycle analysis and induction of apoptosis in
melanoma cells. A) Cell cycle analysis after treatment with QW-296 (free drug and
loaded nanoparticles) to A375 cells, and B) B16F10 cells. C) Effect of QW-296
treatment on the expression of G2/M related proteins PTTG and Cyclin B1. D) Induction
of apoptosis by QW-296 as free drug and loaded in nanoparticles in A375 cells and E)
B16F10 cells after 24 h of incubation. *, p<0.05 (comparison between control vs free
drug), #, p<0.05 (comparison between control vs NP), †, p<0.05 (comparison between
free QW-296 vs QW-296 loaded NP), ns, p>0.05.

ACCEPTED MANUSCRIPT
Poly(ADP-ribose) polymerase (PARP) helps cells to maintain their viability, but its
cleavage facilitates cellular disassembly and serves as a marker of cells undergoing
38
apoptosis.
Therefore, we measured PARP and cleaved caspase 3 by Western blot
analysis after treating A375 and B16F10 cells with QW-296 formulated in the cosolvent
system or loaded into nanoparticles. Treatment of A375 cells with QW-296 resulted in
increased expression of cleaved caspase 3 and subsequent PARP cleavage (Figure
5
A). Similarly, increased expression of cleaved caspase 3 was observed when B16F10
cells were treated with QW-296 formulated in cosolvent or into nanoparticles (Figure
B). These observations provide a reasonable mechanistic explanation of the observed
5
cell death induction by treatment with QW-296.
A A375 cells
QW-296FD (nM)
40 60
QW-296NP (nM)
40
60
8
6
4
2
00
00
00
00
0
3
2
00
50
0
0
Cleaved PARP in A375 cells
Cleaved Caspase 3 in A375 cells
Total PARP
200
1
50
00
50
0
Cleaved PARP
1
Cleaved
Caspase 3
GAPDH
0
40
60
0
40
60
0
40
60
0
40
60
QW-296FD (nM) QW-296NP (nM)
QW-296FD (nM) QW-296NP (nM)
B B16F10 cells
3
00
50
00
50
QW-296FD (nM)
QW-296NP (nM)
40
60
250
Total PARP in B16F10 Cells
Cleaved Caspase 3 in B16F10 cells
2
2
1
0
40
60
0
2
1
1
00
50
00
Total PARP
Cleaved
Caspase 3
100
50
0
5
0
β-Actin
0
0
40
60
0
40
60
0
40
60
0
40
60
QW-296FD (nM) QW-296NP (nM)
QW-296FD (nM) QW-296NP (nM)
Figure 5. Estimation of apoptotic protein expression (PARP and Cleaved Caspase 3) by
WB in A) A375 cells and B) B16F10 cells after treatment with QW-296 in cosolvent and
QW-296 loaded nanoparticles after 48 h of treatment.
Effect of QW-296 on cell migration

ACCEPTED MANUSCRIPT
To determine the effect of QW-296 on cell migration through microtubule
destabilization, we performed a scratch migration assay. After 24 hours, the vehicle
(DMSO) treated A375 and B16F10 cells had nearly achieved complete closure of the
wound by migrating to the scratch area. In contrast, QW-296 inhibited of cell migration
and equivalent to colchicine (Figure 6A). These results demonstrate that QW-296
strongly reduces aberrant cancer cell proliferation and hinders cell migration efficiently
as colchicine in metastatic melanoma. We also estimated the levels of vimentin in A375
cells after treatment with different concentrations of colchicine and QW-296. There was
a significant decrease in vimentin protein expression level in dose dependent manner
after treatment with QW-296 compared to control group. (Figure 6B).

ACCEPTED MANUSCRIPT
Col 60 (nM)
QW-296 60 (nM)
DMSO
A
B
Colchicine (nM) QW-296 (nM)
40 60
40
60
DMSO
Vimentin
GAPDH
Figure 6. Effect of QW-296 and colchicine on cell migration and EMT marker. A)
Scratch assay in B16F10 and A375 cells. B) Dose-dependent decrease in vimentin in
A375 cells.
Effects of QW-296 on taxane-resistant cells
Paclitaxel (PTX) and vinblastine are known to develop chemoresistance to
39,40
24
cancer cells.
ABI-III showed high potency for TXR cell lines. Since ABI-III and QW-
2
96 belong to the same family of compounds, we hypothesized that QW-296 would
retain its ability to circumvent TXR mechanisms. To support this, we screened QW-296
against TXR cell line, such as PC3-TXR. QW-296 was found to be equipotent in both

ACCEPTED MANUSCRIPT
PC3 and PC3-TXR cells with resistance index only 1.3, whereas PTX showed
significantly low potency in PC3-TXR cells with a resistance index of 408.6.
Interestingly, the parent compound ABI-III also showed high potency in resistant lines
with resistance Index of 7.4 (Figure 7A).
Effect of QW-296 on Pgp activity
To identify the potential mechanism for low resistance index of QW-296 in TXR
TM
cells, we determined the effect of QW-296 on Pgp-ATPase activity using Pgp-Glo
assay system. In this assay, a specific amount of ATP was incubated with recombinant
human P-gp and then unmetabolized ATP was detected as a luciferase-generated
luminescent signal. The decrease in luminescence signal after drug treatment reflect
ATP consumption by Pgp. Changes in the luminescence of QW-296 treated samples
from Na3VO4 treated samples were plotted to illustrate the stimulation or inhibition of P-
gp ATPase activity due to QW-296 treatment (Figure 7B). Verapamil, a well-known P-
gp substrate, stimulated P-gp ATPase activity, resulting in significantly decreased
luminescence. Although there was no statistically significant difference in luminescence
changes between the vehicle control treated group and QW-296 treated groups at 10
and 100 nM concentrations, suggesting that QW-296 is a mild stimulator of P-gp
ATPase. However, at high concentration (1000 nM) QW-296 exhibited effects like that
of an activator of Pgp. These results strongly indicate that QW-296 like verapamil, is a
substrate of P-gp and leads to the inhibition of P-gp efflux. This may partially explain the
mechanism of QW-296 to overcoming TXR.

ACCEPTED MANUSCRIPT
A
Entry
IC50 SD (nM, n = 3)
-
-
Paclitaxel
.2ꢁ0.64
716.6ꢁ76.38
08.6
ABI-III
QW-296
4
15ꢁ5
190ꢁ20
PC3
1
111ꢁ18.93
243.3ꢁ15.28
PC3-TXR
4
7.4
1.3
*RI
B
1
1
1
1
1
800
600
400
200
000
ns
*
8
6
4
2
00
00
00
00
0
NT
Verapamil
10
100
1000
QW-296 (nM)
Figure 7. Determination of resistance index and possible mechanism to overcome
chemoresistance by QW-296. A) Anti-proliferative activity of paclitaxel (PTX), ABI-III
and QW-296 on paclitaxel-resistant PC3-TXR cells and non-resistant PC3 cells. B)
Effect of QW-296 on Pgp ATPase activity. Change in luminescence compared to 100
μM Na3VO4 treated samples was plotted (mean ± SD, n = 3). QW-296 at lower
concentrations (10 and 100 nM) showed a non-significant effect compared to NT control
on Pgp ATPase activity. *, p < 0.05.
In vivo efficacy of QW-296 loaded nanoparticles
Preclinical efficacy of QW-296 loaded nanoparticles was evaluated in a lung metastatic
melanoma model generated by intravenous injection of B16-F10-Luc cells into C57BL/6
6
2
albino mice. The mice with the bioluminescent radiance of 10 p/sec/cm /sr were
randomized into three groups: 1) control, 2) QW-296 free drug treatment group, and 3)
QW-296 loaded nanoparticles treatment group. Mice treated with QW-296 formulated in
cosolvent or loaded into nanoparticles showed inhibition of tumor growth compared to