Telodendrimer-Based Macromolecular Drug Design using 1,3-Dipolar Cycloaddition for Applications in Biology

Article abstract of DOI:10.3390/molecules25040857

An architectural polymer containing hydrophobic isoxazole-based dendron and hydrophilic polyethylene glycol linear tail is prepared by a combination of the robust ZnCl₂ catalyzed alkyne-nitrile oxide 1,3-dipolar cycloaddition and esterification chemistry. This water soluble amphiphilic telodendrimer acts as a macromolecular biologically active agent and shows concentration dependent reduction of glioblastoma (U251) cell survival.

Full text of DOI:10.3390/molecules25040857

molecules
Article
Telodendrimer-Based Macromolecular Drug Design
using 1,3-Dipolar Cycloaddition for Applications
in Biology
Hossein Yazdani 1,2, Esha Kaul 3, Ayoob Bazgir 2, Dusica Maysinger 3,
*
and Ashok Kakkar 1,
*
1
Department of Chemistry, McGill University, 801 Sherbrooke St. West, Montréal, QC H3A 0B8, Canada;
hyazdani21@gmail.com
Department of Chemistry, Shahid Beheshti University G.C., Tehran 1983963113, Iran; a_bazgir@sbu.ac.ir
Department of Pharmacology and Therapeutics, McGill University, 3655 Promenade Sir William Osler,
Montréal, QC H3G 1Y6, Canada; esha.kaul@mail.mcgill.ca
2
3
*
Correspondence: dusica.maysinger@mcgill.ca (D.M.); ashok.kakkar@mcgill.ca (A.K.);
Tel.: +1-514-398-6912 (A.K.)
ꢀꢁꢂꢀꢃꢄꢅꢆꢇ
ꢀꢁꢂꢃꢄꢅꢆ
Academic Editor: Jørn Bolstad Christensen
Received: 21 January 2020; Accepted: 12 February 2020; Published: 15 February 2020
Abstract: An architectural polymer containing hydrophobic isoxazole-based dendron and hydrophilic
polyethylene glycol linear tail is prepared by a combination of the robust ZnCl2 catalyzed
alkyne-nitrile oxide 1,3-dipolar cycloaddition and esterification chemistry. This water soluble
amphiphilic telodendrimer acts as a macromolecular biologically active agent and shows concentration
dependent reduction of glioblastoma (U251) cell survival.
Keywords: telodendrimer; macromolecular drug; 1,3-dipolar cycloaddition; glioblastoma
1. Introduction
The isoxazole moiety, a five-membered heterocycle containing oxygen and nitrogen at adjacent
positions, has shown interesting biological properties including anticancer [
antibacterial [ ], anti-inflammatory [ ], antiviral [ ], antithrombic [ ], antiapoptotic [
antiparasitic activity [ ]. For example, NVP-AUY922 (Figure 1A) is an anticancer agent [
1
], antimicrobial [
] and
] and
1],
2
3
4
5
6
1
7
thiophen-2-yl isoxazole (Figure 1B) significantly inhibits proliferation of HeLa cells. Isoxazole with
a steroid moiety (Figure 1C) has displayed considerable cytotoxicity towards tumor cell lines
and sulfisoxazole, flucloxacillin, dicloxacillin, oxacillin, sulfamethoxazole and cloxacillin are some
examples of marketed antimicrobial drugs bearing an isoxazole ring [
nonsteroidal anti-inflammatory drug (NSAID) that was targeted towards cyclooxygenase enzymes
to hinder prostaglandin biosynthesis and to reduce pain [ ]. About thirty patents covering different
isoxazole-based structures have been reported showing a wide array of chemical modifications and
applications of such compounds [ 18]. Macromolecular therapeutics are increasingly proposed as
8]. Valdecoxib (Figure 1D) is a
8
8
–
nanomedicines [19] and new methodologies to prepare highly potent isoxazoles bearing nanocarriers
and nanotherapeutics will likely expand the scope of their applications.
Molecules 2020, 25, 857; doi:10.3390/molecules25040857
www.mdpi.com/journal/molecules

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HO
O
S
H
H
N
O
O
H
N
N
HO
N
H
O
S
O
O
O
HO
N
S
O
NH2
Ph
O
N
O
A
B
C
D
Figure 1. Selected examples of bioactive isoxazoles:
A
,
B
and C, 3,4,5-isoxazole and fused isoxazole
derivatives with anticancer; and D, 3,4,5-isoxazole derivative with anti-inflammatory activity.
A tremendous research effort has been devoted to developing nanocarriers that could deliver
active pharmaceutical agents to the sites affected by pathological changes to minimize wide body
distribution and reduce undesirable side effects [20,21]. Amphiphilic linear block copolymers have been
extensively explored in assembling core-corona type spherical micelle structures for drug delivery [22].
It is now well known that the overall macromolecular composition and structure play a vital role
in modulating important parameters including plasma residence time and drug loading/controlled
release behavior in these formulations [23]. This has led to expanding the macromolecular space
from linear-to-branched-to-hyperbranched architectures [24]. Telodendrimers that are hybrids of
hyperbranched dendrimers and linear polymers have offered an advantageous platform in combining
individual properties of these components [25–29].
Considering the biological potency of isoxazoles, we were interested in developing synthetic
methodologies to water-soluble macromolecular drugs using amphiphilic architectural polymers,
in which the hydrophobic dendron segment will contain isoxazole heterocyclic rings and a linear
poly(ethylene glycol) methyl ether (mPEG) tail will impart hydrophilic character. 1,3-Dipolar
cycloaddition is among some of the most important reactions in organic and medicinal chemistry [30
]
and it has been used for the isoxazoles scaffold synthesis by the cycloaddition of nitrile oxides and
alkynes [31]. To the best of our knowledge, employing 1,3-dipolar cycloaddition reaction based on
nitrile oxides in synthesizing dendrons/dendrimers has not been reported. We report herein such a
synthetic approach to construct an isoxazole-mPEG based architecture polymer, its behavior in an
aqueous medium and an assessment of its ability to act as a macromolecular agent that can reduce or
even abolish viability of glioblastoma cells.
2. Results and Discussion
2.1. Synthesis
We first explored the potential of 1,3-dipolar cycloaddition strategy in the synthesis of a symmetric
generation 0 dendrimer. It was begun by the propargylation of pentaerythritol using propargyl bromide
with potassium hydroxide in dry DMF (Scheme 1) [32], followed by the 1,3-dipolar cycloaddition
◦
on the tetrapropargylated core (
3
) using a bromonitrile oxide precursor (
4
) in THF at 45 C for
3 h, with NaHCO3 as base and ZnCl2 as a catalyst. It afforded 5,50-(((2,2-bis(((3-bromoisoxazol-
5-yl)methoxy)methyl)propane-1,3-diyl)bis(oxy))bis(methylene))bis(3-bromoisoxazole) (
isolated yield (Scheme 1) [33].
5) in 44%

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O
O
O
O
OH
OH
OH
KOH
Br
+
dry DMF
HO
2
3
1
OH
N
Br
Br
THF, NaHCO3
ZnCl2 10%mol
4
Br
N
O
O
N
O
O
O
O
Br
Br
N
O
O
N
5
Br
Scheme 1.
Synthesis of 5,50-(((2,2-bis(((3-bromoisoxazol-5-yl)methoxy)methyl)propane-1,3-
diyl)bis(oxy))bis(methylene))bis(3-bromoisoxazole).
Isoxazole bearing dendrimer
5 was not soluble in common organic solvents and it prevented
us from expanding the scope of dendrimer build-up or its surface functionalization to enhance its
solubility and examine its biological activity. We subsequently considered designing an architectural
polymer by replacing one of the isoxazole based arm with PEG. For this proposal, we first synthesized
a tripropargylated pentaerythritol core by reacting pentaerythritol with 3 equivalents of propargyl
bromide [28], which leaves a free OH group at the core (
by reacting mPEG2000 with succinic anhydride in the presence of triethylamine (Et3N) in CHCl3 at
70 ◦C for 24 h. The esterification reaction of mPEG-COOH (
) with the tripropargylated pentaerythritol
core ( ) was subsequently carried out using 1,4-dimethylpyridinium p-toluenesulfonate (DPTS) and
6, Scheme 2). mPEG-COOH (9) was synthesized
9
6
diisopropylcarbodiimide (DIPC) in dry CH2Cl2. The desired telodendrimer with isoxazole rings was
subsequently synthesized using a 1,3-dipolar cycloaddition reaction on 10, using a bromonitrile oxide
precursor (Scheme 2).
Telodendrimer 11 was soluble in common organic solvents and it was characterized using a
combination of techniques including nuclear magnetic resonance (NMR) and mass spectrometry.
Its aqueous behavior was first examined using 1H NMR in D2O (Figure 2). As expected, dendron peaks
become broad while those related to polyethylene glycol retained their original peak configurations.
This suggests that the telodendrimer assumes a hydrophobic core/hydrophilic corona type structure
in D2O.

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O
O
O
OH
OH
OH
KOH
Br
+
dry DMF
HO
HO
2
6
1
O
O
CHCl3, Et3N
70 oC, 24h
O
O
OH
O
OH
O
n
n
O
n= 45
O
9
7
8
DIPC, DPTS
DCM, N2, 24h
6
Br
N
O
O
OH
N
N
O
O
O
O
O
O
Br
O
O
Br
Br
4
O
O
O
O
O
O
n
THF, NaHCO3
ZnCl2, 45 o
n
O
O
C
O
10
11
N
Br
Scheme 2. Synthesis of isoxazole-mPEG architectural polymer (11).
Figure 2. 1H nuclear magnetic resonance (NMR) (500 MHz) spectrum of isoxazole telodendrimer in
D2O (A) and CDCl3 (B).
We examined similar self-assembly of the telodendrimer using an acetone/water co-solvent
evaporation method [34–36]. The resulting assemblies were characterized using dynamic light
scattering (DLS) and transmission electron microscopy (TEM) (Figure 3). These suggested that
spherical structures were formed with a size distribution of 200 and ~60 nm, as measured by DLS and
TEM respectively. As is common with solution based DLS measurements, hydrodynamic sizes of these

Molecules 2020, 25, 857
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assemblies were found to be higher as compared to TEM [37]. We were unable to obtain a critical
micelle concentration of the assembled structures using pyrene encapsulation. This may suggest that
pyrene does not get incorporated into the hydrophobic core due to inability of the self-assembled
structures from these telodendrimers to solubilize pyrene [38]. However, using the DLS intensity
method, an inflection point was measured to be at 0.35 mg/mL. This value is high and suggests
that at dilutions encountered during circulation, the particles observed through DLS and TEM will
be disassembled.
120
100
80
60
40
20
0
0
200
400
600
800
1000
Diameter (nm)
(A)
(B)
Figure 3.
(A
) Dynamic light scattering (DLS) size distribution and (
B
) transmission electron microscopy
(TEM) image for the aqueous assemblies of isoxazole telodenderimer. The concentration of the
polymeric micelle solution was 2 mg/mL for TEM study.
2.2. Isoxazole Telodendrimer Reduces Glioblastoma Cell Viability
We explored the concentration-dependent cytotoxicity of the telodendrimer using cell viability
assay. Brain tumor cells (U251N) were treated with increasing concentrations of the telodendrimer,
ranging from 1 nM to 100 µM and cell nuclei were counted after 24 h and 72 h. As an additional
control, cells were treated with 15% milli-q (MQ) water, since the telodendrimer was diluted in milli-q
water to make stock concentrations. The presence of MQ water did not affect cell viability. None of
the concentrations below 10
µM caused significant cell death after 24 h and 72 h (Figure 4). However,
100 M concentration of the telodendrimer significantly reduced the number of viable cells to 9.89%
µ
and 1.35% after 24 h and 72 h, respectively (Figure 4). Results from this study demonstrate that
isoxazole-based telodendrimer acts as a macromolecular drug and exhibits good cytotoxicity towards
U251N cells. Isoxazole based compounds, such as NVP-AUY922 [39], as well as Leflunomide [40
]
exhibit anti-tumor activity. Certain 3,5-diaryl isoxazole linked 2,3-dihydroquinazolinones are cytotoxic
towards U251N cells. These compounds kill cells by disrupting microtubules and fragmenting
cell nuclei [41]. Morphological abnormalities seen in U251 glioblastoma cells exposed to the
telodendrimer included pyknotic nuclei, suggesting a small contribution of apoptotic cell death.
Analysis of mitochondrial functions in these cells (using tetrazolium salt, an indicator of mitochondrial
metabolic activity) showed reduced function of these organelles upon telodendrimer treatment.
Mitochondrial functional impairment was also concentration dependent; it was most profound in
> 10 µM telodendrimer concentrations and it correlated with the decline in glioblastoma cell number.
We are currently investigating several modes of cell death caused by isoxazole-based telodendrimers
including ferroptosis [42
–50]. Our further aim in this direction is to explore cell death mechanism,
to identify the implicated signal transduction pathways.

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Figure 4. Glioblastoma cell viability studies: U251N cells were treated with aqueous formulations
of the telodendrimer (1 nM, 1 M, 10 M, 25 M, 50 M and 100 M) for up to 72 h. Cells were
µ
µ
µ
µ
µ
fixed and stained with Hoechst 33342. Vehicle indicates 15% milli-q water. More than 130 images
were analyzed for each condition. Number of cells were quantified by imaging them using Leica
Fluorescent Microscope.
3. Materials and Methods
3.1. General
The NMR spectra were recorded on AV 400 and 500 MHz (Bruker, Beerlika, MA, USA)
spectrometers both equipped with BBFO+ smart probes. Mass spectra analyses (HRMS, ESI) were
performed on an Exactive Plus Orbitrap-API (Thermo Scientific, Waltham, MA, USA) high resolution
mass spectrometer. Chemicals were purchased from Sigma Aldrich (St. Louis, MO, USA), ChemImpex
International (Wood Dale, IL, USA), Alfa Aesar (Ward Hill, MA, USA), Fisher scientific (Hampton, NH,
USA) and ACP Chemicals (Saint-Leonard, QC, Canada), and were used without further purification.
Dry solvents were obtained from a solvent purification system. TEM samples for imaging were
prepared by placing a 10
µL drop of the polymeric micelle solution (2 mg/mL) onto a carbon-coated
copper grid. Excess solution was removed after 2 min using a Whatman filter paper. 10
µL of 2%
uranyl acetate was then dropped onto the grid, left for 2 min and excess removed by a Whatman filter
paper as before. The grid was then left to dry for an additional 20 min before imaging.

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3.2. Synthesis
3-(3-(prop-2-yn-1-yloxy)-2,2-bis((prop-2-yn-1-yloxy)methyl)propoxy)prop-1-yne
A mixture of pentaerythritol (1.00 g, 7.35 mmol) and potassium hydroxide (6.25 g, 0.112 mol) in
dry DMF (25 mL) was stirred at 0 ◦C under nitrogen. After 5 min a solution of propargyl bromide
(7.8 mL, 0.084 mol) in toluene (80%) was added drop wise and the reaction mixture was left to stir for
24 h. After completion of the reaction, water was added, and the compound was extracted with diethyl
ether (3
×
5 mL) and washed with brine. The organic layer was isolated and dried over anhydrous
Na2SO4. The solvent was removed under vacuum and the desired product was purified by column
1
chromatography (hexane/EtOAc = 8.5:1.5) (Yield: 1.2 g, 57%). H NMR (400 MHz, CDCl3): δH (ppm)
2.41 (4H, t, 4JHH = 2.4 Hz, C
≡
CH), 3.52 (8H, s, C-CH2-O), 4.12 (8H, d, 4JHH = 2.4 Hz, O-CH2-C
C).
≡
13C{1H} NMR (100 MHz, CDCl3): δC (ppm) 44.8, 58.7, 69.0, 74.0, 80.0.
5,50-(((2,2-bis(((3-bromoisoxazol-5-yl)methoxy)methyl)propane-1,3-diyl)bis(oxy))bis(methylene))bis(3-
bromoisoxazole).
A mixture of tetrakis(2-propynyloxymethyl) methane (0.288 g, 0.001 mol), dibromoformaldoxime
(1.010 g, 0.005 mol), ZnCl2 (40 mol%) and NaHCO3 (0.672 g, 0.008 mol) in THF (5 mL) was stirred at
45 ◦C for 3 h. The solvent was then removed under vacuum and the crude mixture was washed with
H2O (10 mL), diethyl ether (10 mL) and then dried to afford the desired pure product (Yield: 0.340 g,
1
44%). H NMR (400 MHz, DMSO-d6): δH (ppm) 3.43 (8H, s, C-CH2-O), 4.60 (8H, s, O-CH2-isoxazole
ring), 6.76 (4H, s, HC=C).
3-(prop-2-yn-1-yloxy)-2,2-bis((prop-2-yn-1-yloxy)methyl)propan-1-ol
A mixture of pentaerythritol (1.00 g, 7.35 mmol) and potassium hydroxide (2.8 g, 0.05 mol) in
dry dimethylformamide (DMF, 20 mL) was stirred at 0 ◦C under nitrogen. After 5 min a solution of
propargyl bromide (4.76 g, 5.95 mL, 0.040 mol) in toluene (80%) was added drop wise to the reaction
mixture. After warming to room temperature slowly, the reaction mixture was left to stir for 24 h at
40 ◦C under nitrogen. After completion of the reaction and cooling to room temperature, water was
added to the mixture and the compound extracted with diethyl ether (3
The organic layers were isolated and dried over anhydrous Na2SO4. The solvent was removed under
×
5 mL) and washed with brine.
vacuum. Purification of the crude material was done by column chromatography (hexane/EtOAc =

Molecules 2020, 25, 857
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1
7.5:2.5) to afford the desired product (Yield: 1.1 g, 60%). H NMR (500 MHz, CDCl3): δH (ppm) 2.43
(3H, t, 4JHH = 2.5 Hz, C CH), 3.56 (6H, s, C-CH2-O), 3.68 (2H, s, CH2-OH), 4.12 (6H, d,4JHH = 2.5 Hz,
≡
O-CH2-C≡C). 13C{1H} NMR (75 MHz, CDCl3): δC (ppm) 44.6, 58.7, 64.9, 70.1, 74.5, 79.6.
mPEG2000-COOH
A mixture of poly(ethylene glycol) methyl ether 2000 (mPEG 2000) (3g, 1.5 mmol), succinic
anhydride (0.25 g, 0.002 mol) and Et3N (0.2 ml, 0.0015 mol) in CHCl3 (5 mL), was refluxed at 70 ◦C
for 24 h. After completion of the reaction, water was added to the mixture and the compound was
extracted (3
×
10 mL) with chloroform and washed with brine. The organic layer was isolated and
dried over anhydrous Na2SO4. The solvent was then removed under reduced pressure and dried.
The solid residue was purified further from CHCl3/Ether (1:5) to afford the pure product with a yield of
1
2.7 g, 86%. H NMR (400 MHz, CDCl3): δH (ppm) 2.66 (4H, m, -CH2-CH2-COOH), 3.40 (3H, s, OCH3),
3.11–3.85 (178 H, m, PEG), 4.28 (2H, t, 3JHH = 4 Hz, -CH2-OCO-). 13C{1H} NMR (125 MHz, CDCl3): δC
(ppm) 29.4, 44.9, 58.9, 63.6, 68.9, 70.5, 71.8, 172.4, 174.6.
mPEG2000-tripropargyl pentaerythritol
In a three-neck flask mPEG-COOH (2.1 g, 0.001 mol), tripropargyl pentaerythritol (0.25 g, 0.001 mol)
and DPTS (0.279 g, 0.001 mol) were dissolved in dry CH2Cl2. A solution of diisopropylcarbodiimide
(DIPC 0.176 g, 0.0014 mol) in dry CH2Cl2 was added dropwise to the flask over 1.5 h under nitrogen
atmosphere. The reaction was stirred for 24 h. Water was then added and the compound extracted
(2 × 10 mL) with dichloromethane. The mixture was then washed with NaHCO3 (5%) and extracted
with CH2Cl2. After that, the organic layer was washed with brine and then dried over anhydrous
Na2SO4. The solvent was removed under reduced pressure. Purification of the crude material was
1
done by column chromatography (CH2Cl2/methanol = 10:1) (Yield: 0.750 g, 32%). H NMR (500 MHz,
CDCl3): δH (ppm) 2.46 (3H, t, 4JHH = 2.4 Hz, C
≡
CH), 2.67 (4H, m, O=C-CH2-CH2-C=O), 3.40 (3H, s,
OCH3), 3.66 (180H PEG and 8H C-CH2-O, m), 4.13 (6H, d, 4JHH = 2.4 Hz, O-CH2-C C). 13C{1H} NMR
≡
(75 MHz, CDCl3): δC (ppm) 29.0, 29.1, 44.0, 63.6, 63.8, 68.6, 68.9, 69.0, 70.5, 71.9, 74.4, 79.7, 171.7, 172.2.
mPEG2000-isoxazole
A mixture of mPEG-tripropargylpentaerythritol (0.5 g, 0.00021 mol), ZnCl2 (30 mol%) and NaHCO3
(0.1 g, 0.00126 mol) in THF (5 mL) was stirred at 45 ◦C under nitrogen overnight. After completion of
the reaction, the solvent was removed under reduced pressure. The crude was extracted three times
with CH2Cl2. After that the organic layer was washed with brine and then dried over anhydrous
Na2SO4. The solid residue after removing solvent in vacuo was purified using a mixture of CHCl3/Ether

Molecules 2020, 25, 857
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1
(1:5) to afford the desired pure product with a yield of 0.3 g, 52%. H NMR (500 MHz, CDCl3): δH
(ppm)2.60 (2H, t, 3JHH = 5 Hz, O=C-CH2-CH2-C=O), 2.67 (2H, t, 3JHH = 5 Hz, O=C-CH2-CH2-C=O),
3.40 (3H, s, OCH3), 3.48-4.25 (PEG and 8H C-CH2-O, m), 4.57 (6H, s, O-CH2-isoxazole ring), 6.36 (3H,
s, HC=C). 13C{1H} NMR (100 MHz, CDCl3): δC (ppm) 28.9, 44.6, 59.0, 62.5, 63.91, 63.94, 69.0, 70.1,
1
70.3, 70.4, 71.8, 77.2, 106.6, 140.4, 170.8, 171.9, 172.3. H NMR (500 MHz, D2O): δH (ppm) 2.56 (4H,
broad, O=C-CH2-CH2-C=O), 3.30 (3H, s, OCH3), 3.20-4.14 (PEG and 8H C-CH2-O, m), 4.50 (6H, broad,
O-CH2-isoxazole ring), 6.43 (3H, broad, HC=C). ESI-HRMS: m/z = 2710 [M + Na+].
3.3. Methods
Telodendrimer stock solutions were prepared in MilliQ water (500
µM) and polymer itself was
dissolved in DMSO. Different concentrations (1 nM, 1 M, 10 M, 15 M, 25
µ
µ
µ
µ
M, 50 M and 100 M)
µ
µ
were prepared by serial dilutions and used to treat cells as described below.
3.4. Cell Culture
Human U251N GBM cells were originally obtained from the American Type Culture Collection
(ATCC; Rockville, MD, USA). They were cultured in Dulbecco’s Modified Eagle Medium (DMEM;
Gibco, Life Technologies Inc. Burlington, ON, Canada) with 5% (v/v) fetal bovine serum (FBS; Gibco)
and 1% penicillin-streptomycin (P/S; Gibco). Cells were incubated at 37 ◦C with 5% CO2 and 95%
relative humidity.
3.5. Cell Viability Assay
U251N GBM cells were seeded in 96 well plates (5000 cells/well) for 24 h. They were then exposed
to different concentrations of the telodendrimer (1 nM, 1
for 24 h or 72 h before being fixed with 4% paraformaldehyde (PFA). The fixed cells were then washed
with phosphate buffered saline (PBS) and incubated with 10 M Hoechst 33342 (Invitrogen, Eugene,
µM, 10 µM, 15 µM, 25 µM, 50 µM and 100 µM)
µ
OR, USA) for 10 min. This solution was aspirated and replaced with PBS. The cells were then imaged
using Leica Fluorescence Microscope (DMI4000B). Images for each condition were analyzed using
ImageJ. Cell viability was measured by cell counting. At least one hundred cells were analyzed in
9 random fields per cover slip per condition. Three independent experiments were performed. The cell
loss was expressed as % relative to untreated or vehicle (DMSO) treated controls.
3.6. Statistics
All data are expressed as mean
for statistical analyses. P-values less than 0.05 were considered significant. Microsoft Excel’s inbuilt
statistical functions were used for all calculations.
±
SEM (Standard Error of Mean). Student’s t-tests were used
4. Conclusions
A simple synthetic methodology based on the 1,3-dipolar cycloaddition reaction between an
alkyne based core and a bromonitrile oxide, followed by esterification for a stitching hydrophilic
polyethylene glycol tail to the core, provides a novel route to architectural polymers containing an
isoxazole-based dendron. The addition of a polyethylene glycol tail to the telodendrimer confers
solubility in a variety of solvents including aqueous medium, as well as steric stability to the
telodendrimer structure [51,52]. These architectural polymers are candidate macromolecular agents
exerting marked loss of notoriously resistant glioblastoma cells to therapeutic interventions and our
results show that U251N glioblastoma cells are almost completely eliminated when exposed to the
telodendrimer. Considering that combination therapy has been proven to be superior to monotherapy
for cancer treatment [53], different anti-cancer drugs together with this telodendrimer [25,28] merit
further investigations. Certain nanotherapeutics such as liposomal doxorubicin [54] and PTX-loaded
human serum albumin nanoaggregates [55] are Food and Drug Administration (FDA)-approved cancer

Molecules 2020, 25, 857
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nanotherapeutics. However, their relatively large size (130 nm diameter) restricts tissue penetration
and retention. In contrast, telodendrimer based nanoformulations have a smaller size and could
potentially be alternative combination anticancer nanotherapeutics. The new methodology presented
here provides a versatile platform to expand the scope of isoxazole based systems in nanomedicine.
The dendron-linear polymer space in these architectural polymers could be tuned for desired core-corona
type assemblies with low critical micelle concentrations. Their potential for combination therapies
needs to be further explored by covalent and non-covalent linking of anti-tumor agents, as well as the
incorporation of cell surface binding molecular probes for an improved cell-type specificity.
Author Contributions: A.K. and H.Y. conceived the project. A.K., H.Y. and A.B. designed experiments. D.M.
provided biological background and advising for experimental design. H.Y. synthesized and characterized
telodendrimer. E.K. provided data for Figure 4. H.Y., A.K. and D.M. contributed to writing the manuscript. A.K.,
D.M., H.Y. and A.B. edited and revised the manuscript. All authors have read and agreed to the published version
of the manuscript.
Funding: This research was funded by Natural Sciences and Engineering Research Council of Canada (AK:
RGPIN 2018-05610, and DM: RGPIN 04994-15) and Canadian Institute of Health Research (DM: MOP-119425).
Acknowledgments: We gratefully acknowledge the financial support from Natural Sciences and Engineering
Research Council of Canada and the Quebec Center for Advanced Materials/The Center Québécois sur les
matériaux fonctionnels (QCAM/CQMF), the Research Council of Shahid Beheshti University and the Iran National
Science Foundation (INSF).
Conflicts of Interest: The authors declare no conflict of interest.
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