Synthetic copolymer conjugates of docetaxel and: In vitro assessment of anticancer efficacy

Article abstract of DOI:10.1039/d0nj03425h

Docetaxel (DTX) is a widely used chemotherapy drug that is associated with numerous side effects and limited bioavailability. Macromolecular conjugates of DTX may improve drug targeting, solubility, reduce off-target toxicity, and overcome mechanisms of m

Full text of DOI:10.1039/d0nj03425h

View Article Online
View Journal
NJC
New Journal of Chemistry
Accepted Manuscript
A journal for new directions in chemistry
This article can be cited before page numbers have been issued, to do this please use: C. W. Evans, S.
Edwards, J. A. Kretzmann, G. Nealon, R. Singh, T. Clemons, M. Norret, C. Boyer and S. Iyer, New J. Chem.,
2020, DOI: 10.1039/D0NJ03425H.
Volume 42
This is an Accepted Manuscript, which has been through the
Number
6
21 March 2018
Pages 3963-4776
Royal Society of Chemistry peer review process and has been
accepted for publication.
NJC
New Journal of Chemistry
rsc.li/njc
A journal for new directions in chemistry
Accepted Manuscripts are published online shortly after acceptance,
before technical editing, formatting and proof reading. Using this free
service, authors can make their results available to the community, in
citable form, before we publish the edited article. We will replace this
Accepted Manuscript with the edited and formatted Advance Article as
soon as it is available.
You can find more information about Accepted Manuscripts in the
Information for Authors.
Please note that technical editing may introduce minor changes to the
text and/or graphics, which may alter content. The journal’s standard
Terms & Conditions and the Ethical guidelines still apply. In no event
shall the Royal Society of Chemistry be held responsible for any errors
or omissions in this Accepted Manuscript or any consequences arising
from the use of any information it contains.
ISSN 1144-2546
PAPER
Chechia Hu, Tzu-Jen Lin et al.
Yellowish and blue luminescent graphene oxide quantum dots
prepared via a microwave-assisted hydrothermal route using
H2O2 and KMnO4 as oxidizing agents
rsc.li/njc

Please do not adjust margins
Page 1 of 8
New Journal of Chemistry
1
2
3
4
5
6
7
View Article Online
DOI: 10.1039/D0NJ03425H
ARTICLE
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Synthetic copolymer conjugates of docetaxel and in vitro
assessment of anticancer efficacy
Cameron W. Evans,*,a Sky Edwards,a Jessica A. Kretzmann,a,# Gareth L. Nealon,b Ruhani Singh,a,#
Received 00th January 20xx,
Accepted 00th January 20xx
Tristan D. Clemons,a,# Marck Norret,a Cyrille A. Boyerc and K. Swaminathan Iyer*,a
DOI: 10.1039/x0xx00000x
Docetaxel (DTX) is a widely used chemotherapy drug that is associated with numerous side effects and limited bioavailability.
Macromolecular conjugates of DTX may improve drug targeting, solubility, reduce off-target toxicity, and overcome
mechanisms of multidrug resistance. However, most polymer conjugates of DTX investigated to date make use of
biopolymers, which are of fixed structure and are not well suited to optimisation and subsequent reaction to introduce
further functionality. Here, we show the preparation of synthetic copolymer conjugates of DTX with drug loading of up to
20% w/w that also has potential for tuning backbone hydrophilicity and the number of reactive sites for conjugation. The
intermediates produced are comprehensively characterised, as are the macromolecular conjugates, which are tested in the
MCF-7 human breast adenocarcinoma cell line to assess toxicity and anticancer efficacy. The conjugates produced have IC50
values within one order of magnitude of DTX, as expected for slow release of DTX by ester hydrolysis. The results suggest
that the system is promising for delivery of DTX and future work may examine conjugates of a wider molecular weight range,
optimisation of DTX and PEG conjugation efficiency, and in vivo biodistribution.
formulation containing surfactants and/or a non-polar vehicle
such as Cremophor EL®, which is associated with some adverse
effects.7 Treatment with DTX itself is associated with severe
Introduction
Breast cancer constitutes 12% of the global incidence of cancer,
with over 2 million new cases and more than 600,000 deaths
worldwide in 2018.1 It is the most commonly diagnosed cancer
in women, and the leading cause of cancer-related death.
Although a number of treatment strategies exist for breast
cancer by targeting particular tumour subtypes based on
expression of hormone receptors and Her2, chemotherapeutic
cocktails are still widely used alone or as an adjuvant therapy to
ensure complete tumour removal, particularly for aggressive
subtypes.2,3 Following the success of the parent taxane
paclitaxel, semisynthetic docetaxel (DTX) is now the most
widely used chemotherapeutic drug in the treatment of breast
cancer.4–6 Taxanes have limited aqueous solubility, resulting in
a moderate bioavailability, and are usually administered in a
toxicity and debilitating side effects resulting from a lack of
discrimination and selectivity. DTX is an anti-mitotic drug that
interferes with cell replication by binding to β-tubulin during
mitosis, inhibiting microtubule assembly.8,9 As a result, DTX
induces apoptosis in any dividing cells of the body, especially
those which rapidly divide such as hair follicles, finger nails, and
cells of the immune system. Sensory nerve damage to the
extremities is also a common side effect.10 Additionally, the
development of multidrug resistance as a consequence of
taxane-based therapy, particularly in the metastatic context, is
strongly associated with a poor clinical prognosis.11
Drug conjugation to biocompatible macromolecules has been
shown to help ameliorate the adverse effects of
chemotherapeutic drugs. For example, paclitaxel is presently
available as an albumin conjugate (Abraxane®) which displays
improved efficacy and reduced side effects.12–14 Conjugates of
taxanes with biopolymers such as hyaluronic acid have been
previously reported.15–17 However, such natural polymers are of
fixed structure and are not readily optimised. In addition to
biomacromolecular conjugates, synthetic polymer conjugates
have also shown potential as drug delivery agents. Nanocarriers
have the potential to improve on ‘free’ drugs in a number of
ways. These include: improving bioavailability,18,19 reducing the
rate of drug degradation and/or metabolism, manipulation of
drug pharmacokinetics, on-demand drug release, targeting of
specific cell types, combination with a range of imaging
modalities, and combination therapies that deliver multiple
components simultaneously.20–28 Further, drug release from
a. School of Molecular Sciences, University of Western Australia, 35 Stirling
Highway, Crawley WA 6009, Australia.
b. Centre for Microscopy, Characterisation and Analysis, University of Western
Australia, 35 Stirling Highway, Crawley WA 6009, Australia.
c. Faculty of Engineering, The University of New South Wales, UNSW Sydney, High
Street, Kensington NSW 2052, Australia.
* E-mail: cameron.evans@uwa.edu.au (C.W.E.).
* E-mail: swaminatha.iyer@uwa.edu.au (K.S.I.).
Present Addresses:
# Jessica A. Kretzmann: Laboratory for Biomolecular Nanotechnology, Technical
University of Munich, Am Coulombwall 4a, 85748 Garching, Germany.
# Ruhani Singh: CSIRO Manufacturing, New Horizons Centre, 20 Research Way,
Clayton VIC 3168, Australia.
# Tristan D. Clemons: Simpson Querrey Institute, Northwestern University, 303 E.
Superior Suite 11-131, Chicago, IL 60611, USA.
Electronic Supplementary Information (ESI) available: [details of any supplementary
information available should be included here]. See DOI: 10.1039/x0xx00000x
Please do not adjust margins

Please do not adjust margins
New Journal of Chemistry
Page 2 of 8
ARTICLE
Journal Name
1
2
3
and retention (EPR) effect.30 This could result in better patient
Table 1. Characterisation of copolymer backbones and conjugates prepared in this work.
View Article Online
outcomes and a reduction in observedDsOidI:e1-0e.1ff0e3c9t/sD0fNoJr0t3h4o25sHe
undergoing DTX chemotherapy treatment.22 The principal
requirements for EPR targeting are low interaction with blood
components, a molecular weight greater than 40 kDa, and
particles that can circulate for longer than several hours.31
Important considerations for nanoparticle-based delivery of
therapeutics are highlighted in other recent work. For example,
it is important that the carrier itself does not induce any toxic
effects,32 and that the conjugates are carefully purified to
ensure that there is no residual free drug that would result in a
skewed IC50 value.33–35 These considerations need to be
addressed before animal studies can be attempted.
We reasoned that a synthetic macromolecular conjugate of
DTX that has potential for systematically varying the
hydrophobic/hydrophilic balance of the polymer, loading of
DTX, and also subsequent functionalisation would be a
worthwhile pursuit. Evidence of DTX release from the conjugate
by ester hydrolysis is provided. Cytotoxicity is assessed in the
MCF-7 human mammary adenocarcinoma cell line,
demonstrating that the conjugate maintains inhibitory efficacy
comparable to equivalent concentrations of free DTX, but could
increase the duration over which drug is released.
4
5
6
7
8
9
DTX PEG
wt% wt%
1
ID
w
x
y
z
Mw
Mn PDI ΔMw
3a 0.64 0.36
3b 0.50 0.50
3c 0.51 0.49
3d 0.76 0.24
5d 0.76 0.24
8a 0.64 0.34 0.02
8b 0.50 0.48 0.02
8c 0.51 0.47 0.02
8d 0.76 0.20 0.04
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
8.3
8.3
8.7
20
–
–
–
–
–
–
–
–
–
8.65 6.26 1.38
12.8 8.61 1.49
14.8 10.4 1.43
14.2 9.97 1.54
15.5 10.0 1.55 1.3
28.6 18.9 1.51 20
–
–
–
–
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
21.4 18.1 1.18 8.6
n.d.2 n.d. n.d. n.d.
22.5 13.5 1.67 9.6
8e 0.50 0.48 0.01 0.01 5.0
6.9 22.1 17.8 1.24 9.2
1
Indicative changes in molecular weight ΔMw are calculated relative to the
corresponding unconjugated polymer 3 as this was the most similar to PMMA
standards and therefore the most reliably determined value by GPC.
2 n.d. = not determined.
Results and Discussion
In this work, we describe a novel synthetic polymer conjugate
of DTX with high drug loading ability and a size suitable for
targeting by the EPR effect. We make use of an amphiphilic
random statistical copolymer of 2-hydroxyethyl methacrylate
(HEMA, 1) and glycidyl methacrylate (GMA, 2) which produces
a highly hydrophilic backbone containing reactive groups to
allow for drug attachment and functionalisation. We have
recently reported the synthesis, characterisation, and
biocompatibility of such polymer backbones modified for use in
gene delivery applications, both in vitro and in vivo, where they
are well tolerated.36–38 A number of formulations were
prepared to gauge the effect that drug loading and polymer
backbone composition had on the efficacy of the conjugates
produced.
Scheme 1. Synthesis of DTX–copolymer conjugate. Reaction conditions: (i) CuBr, bpy,
ME-Br, MeOH, 80 °C, 2 h; (ii) NH3(aq), MeOH, TEA, 60 °C, 72 h; (iii) mPEG2000-OTs, MeOH,
r.t., 72 h; (iv) DTX-2ʹ-Suc-NHS, DMF, TEA, r.t., 2 h.
DTX–polymer conjugates were prepared by first synthesizing
conjugates can be optimised to be selective for certain
biological environments, such as the reduced pH often
associated within the tumour microenvironment, resulting in
site specific drug release.29 Conjugation also overcomes
mechanisms of multidrug resistance in which ATP-binding
cassette transporter proteins are overexpressed in the cell
membrane; unlike the free drug, larger conjugates cannot be
expelled through such efflux pumps.2
a
water-soluble, amino-modified poly(HEMA-ran-GMA)
polymer backbone 5 (Scheme 1). A reactive prodrug derivative
of DTX, DTX-2ʹ-hemisuccinate N-hydroxysuccinimide ester
(DTX-2ʹ-Suc-NHS, 7), was conjugated to each of the copolymers
to give DTX–polymer conjugates 8. This system allowed for a
number of variables to be systematically controlled, including
the degree of polymerisation and the composition of the
backbone, which in turn governed the molecular weight,
number of reactive sites available for drug conjugation, and
hydrophilicity. Conjugates of differing molecular weight, drug
loading, and functionalisation were synthesised and
characterised to determine the effect on anticancer efficacy
compared to free DTX (Table 1). A PEGylated conjugate 8e was
also produced by reacting methoxy-PEG tosylate (Mw 2000, 4)
with backbone primary amines.
The conjugation of highly hydrophobic moieties to
a
principally hydrophilic backbone would be expected to result in
macromolecules that either self-assemble (e.g., into micellar or
lamellar structures) or fold to minimise interactions of
hydrophobic cargo with the solution. PEGylation may also help
to improve hydrophilicity. Off-target toxicity may be addressed
by targeting delivery of DTX via the leaky vasculature of
tumours through a process known as the enhanced permeation
2 | J. Name., 2012, 00, 1-3
This journal is © The Royal Society of Chemistry 20xx
Please do not adjust margins

Page 3 of 8
New Journal of Chemistry
Please do not adjust margins
Journal Name
ARTICLE
1
2
3
4
View Article Online
DOI: 10.1039/D0NJ03425H
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Figure 1. Characterisation of DTX derivatives. (a) Numbering scheme used for DTX. (b) 1H NMR of copolymer 3d, aminated copolymer 5d, DTX, DTX-2ʹ-Suc-NHS, and the DTX–polymer
conjugate 8d.
Figure 2. Characterisation of DTX–polymer conjugates. (a) Carr-Purcell-Meiboom-Gill (CPMG) 1H NMR demonstrates successful reaction between DTX-2ʹ-Suc-NHS and aminated
copolymer 5. Strong peak attenuation reveals strong binding (i.e., covalent) of DTX and polymer that is not observed in a simple mixture at the equivalent concentration. Spectra are
shown to scale. (b) The NOESY crosspeak at δH 1.04 ppm (polymer backbone CH2 resonances) and DTX aromatic peaks at δH 7.40 ppm and 7.45 ppm (Ar2) demonstrate conjugation.
These peaks retain the same phase as the diagonal, consistent with a large macromolecule, and are not observed in NOESY NMR spectra of equivalent mixtures of free DTX and
polymer. (c) Determination of hydrodynamic size by dynamic light scattering for DTX–polymer conjugate 8d and PEGylated conjugate 8e. Both conjugates show a propensity for self-
aggregation and/or folding in solution. Zeta potentials agree with expected charge based on free amine (+17 mV) and PEG (-12 mV). (d) Release of highly hydrophobic DTX from
polymer can be demonstrated using CPMG 1H NMR. Signals from covalently bound DTX are broadened and suppressed, whereas free DTX resonances are intense, well resolved and
often noticeably shifted from their bound counterparts. The release of DTX from the polymer by ester hydrolysis was followed over 20 days. Spectra are shown to scale.
1
The strategy for conjugating DTX is the addition of a succinate Successful formation of DTX-2ʹ-Suc was indicated via H NMR
linker at the 2ʹ position (Figure 1a). In the case of paclitaxel, spectroscopy by the loss of the DTX 2ʹ OH peak at δH 3.33 ppm.
esterification at this position is known to increase water Successful DTX-2ʹ-Suc-NHS synthesis (white crystalline solid,
solubility and successfully release the parent drug more so than 85% yield) was demonstrated by a suite of NMR spectroscopic
modification at the C7 hydroxyl group.39 In our hands, DTX-2ʹ- techniques including 1H (Figure 1b), 13C, DEPT135, COSY, HSQC,
hemisuccinate (DTX-2ʹ-Suc) as an intermediate for the synthesis and HMBC and mass spectrometry. Successful reaction was
of DTX-2ʹ-hemisuccinate N-hydroxysuccinimide ester was demonstrated by a shift of the 3ʹ amine (δH 7.42 to 7.87 ppm)
unstable owing to hydrolysis during its purification by reverse- and 2ʹ proton (δH 4.33 to 5.1 ppm) resonances relative to DTX.
phase HPLC. Therefore, once prepared, it was washed, dried The presence of new resonances corresponding to the succinate
and then immediately used to produce the NHS ester. moiety (δH 2.78 and 2.98 ppm) and NHS protons (δH 2.80 ppm)
This journal is © The Royal Society of Chemistry 20xx
J. Name., 2013, 00, 1-3 | 3
Please do not adjust margins

Please do not adjust margins
New Journal of Chemistry
Page 4 of 8
ARTICLE
Journal Name
1
2
3
4
View Article Online
DOI: 10.1039/D0NJ03425H
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Figure 3. Cytotoxicity assessed by IC50 determination in MCF-7 mammary epithelial adenocarcinoma cells using MTS assay. Times in each plot refer to the duration of test
compound incubation, after which the compound was removed and cells were incubated in fresh media until the MTS assay was performed at 72 h. For polymer conjugates, [DTX]
refers to the equivalent free DTX concentration; that is, conjugate concentrations were normalised based on determined DTX loading. (a) DTX as the free drug exhibits a
concentration- and time-dependent toxicity profile; (b) Polymer–DTX conjugates show concentration-dependent cytotoxicity with a higher IC50 value than the free drug, presumably
due to slow and sustained release of DTX by ester hydrolysis; (c) PEGylated polymer–DTX conjugate displays a comparable efficacy to DTX and DTX–polymer conjugates; (d)
representative confocal image of MCF-7 cells receiving no treatment; (e) confocal image of cells treated with DTX–polymer conjugate 8b; (f) confocal image of cells treated with
PEGylated DTX–polymer conjugate 8e. Confocal images are maximum intensity projections, 40×/1.30 oil immersion, red = RBITC, DTX–polymer conjugate; green = phalloidin-iFluor
488, cytoskeleton; blue = Hoechst 34580, nuclei.
were observed as expected.
may depart from measured values owing to different column
Successful conjugation of DTX to copolymers was interactions compared to GPC PMMA standards. The particle
demonstrated by a range of NMR techniques. 1H NMR size as measured by dynamic light scattering (DLS, Figure 2c) is
spectroscopy of the product showed broadening of the DTX consistent with the formation of nanoparticle aggregates which
resonances, which was consistent with its successful are better suited to passive targeting than is the free drug.42 The
incorporation into a large macromolecule (Figure 1b).40 Carr- conjugates overcome the poor aqueous solubility of DTX and
Purcell-Meiboom-Gill (CPMG) NMR spectra of the conjugates eliminate the need for addition of polysorbate 80 and ethanol,
displayed significant attenuation of the DTX resonances which are sources of potential hypersensitivity reactions.43
compared to simple mixtures of DTX and polymer, consistent
DTX release in PBS pH 7.4 + 0.1% v/v Tween 80 at 37 °C (Figure
with covalent incorporation into a macromolecular structure S1) as followed by HPLC gave a first-order release profile with a
(Figure 2a).41 Additionally, conjugation of DTX to the polymer time constant of 12.2 h. Release of DTX from the conjugates was
1
backbone is confirmed by Nuclear Overhauser Effect also detectable by CPMG H NMR spectroscopy performed in
Spectroscopy (NOESY), revealing
a proximal relationship MeOD. On day 1, only broad, attenuated peaks were present,
between the polymer backbone and DTX aromatic groups with no resonances characteristic of free DTX observed,
(Figure 2b) that was not observed in simple mixtures of free DTX consistent with covalent conjugation. By day 20, the sharp
and the polymer. Furthermore, crosspeaks were observed with resonances of the drug molecule had returned, demonstrating
the same phase as the diagonal, consistent with a large DTX release from the conjugate (Figure 2d). 1H NMR
macromolecule. In Diffusion Ordered Spectroscopy (DOSY) spectroscopy also provided evidence for cleavage at the 2ʹ
experiments, free DTX diffused at 1.3 × 10-10 m2 s-1, and succinimidyl ester position to return the 2ʹ OH group of DTX and
polymer 5d diffused at 5.7 × 10-11 m2 s-1 ([DTX] ca. 1.6 mM in thus the active drug (δH 7.44 ppm, br
⟶ 7.39 ppm, t). Here, and
DMSO-d6, 298 K). Under the same conditions, conjugate 8d also in in the case of paclitaxel, the rate of hydrolysis of the
diffused at 2.3 × 10-11 m2 s-1, slower than free DTX or polymer, hemisuccinate ester is considerable and therefore alternative
confirming the formation of
a larger macromolecule linkers with greater stability may be worthy of consideration for
(Supporting Information Figure S2). While GPC of conjugates 8 sustained release.39
demonstrated molecular weight (Mw) increases relative to their
In vitro assessment of the DTX–polymer conjugates half
parent backbones 3, the final molecular weights of conjugates maximal inhibitory concentrations (IC50) in the breast cancer cell
4 | J. Name., 2012, 00, 1-3
This journal is © The Royal Society of Chemistry 20xx
Please do not adjust margins

Page 5 of 8
New Journal of Chemistry
Please do not adjust margins
Journal Name
ARTICLE
1
2
3
4
variation of the backbone Mw in this range did not produce any
observable differences in anticancer activity, although higher
View Article Online
DOI: 10.1039/D0NJ03425H
molecular weights could be investigated. Similarly, PEGylated
conjugate 8e also did not alter the cytotoxicity compared to DTX
(72 h treatment times shown in Figure 3c) but conjugates with
higher PEG content could be assessed. Despite the lack of
observable differences in this study, changes in the in vivo
biodistribution of the conjugates might be expected and this
would be worthy of further investigation. A comparison of IC50
values for each of the experimental treatment times is shown in
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Figure
4 (Supporting Information includes a discussion
comparing the efficacy of conjugates and statistical analysis).
Similarly-sized conjugates with different DTX loading (e.g., 8b
vs. 8d) did not substantially affect conjugate efficacy but as
treatments were normalised to DTX content, the delivered total
mass of conjugate 8d was lower. Solubility may determine the
maximum possible DTX loading but a higher degree of
PEGylation or backbone HEMA content may be further
optimised in this system. For comparison, we also mixed DTX
and unconjugated polymer (sample 5e in Fig. 4) in equivalent
ratios to the conjugated samples and observed no statistical
difference in efficacy versus free DTX.
Our results improve on recent work in several ways. First, the
release of DTX from the conjugate followed first-order kinetics
with little detectable burst release as a result of careful removal
of unbound DTX. Second, we achieved similar IC50 values at
lower DTX loadings.34 Third, the drug carrier (polymer
backbone) showed no toxicity even when incubated in 1000×
excess compared to the IC50 of the drug; this is an improvement
over a micellar system.32
Fluorescently-tagged conjugates were produced by reacting
rhodamine B isothiocyanate (RBITC) with backbone primary
amines in DMF/water at pH 8–9. Confocal microscopy
demonstrated that within 2 h, conjugates had localised within
MCF-7 cells (Figure 3d-f). Due to fewer reactive sites on the
polymer backbone, the conjugate 8e has lower RBITC loading
and therefore appears dimmer than the non-PEGylated
counterpart, conjugate 8b. The PEGylated conjugate 8e showed
a lower uptake after 2 h that may be the result of reduced
membrane adhesion (ζ-potential -12 mV vs +17 mV for the
unPEGylated conjugate).
Figure 4. Comparison of IC50 values for each of the DTX–polymer conjugates 8a-e, an
equivalent mix of unconjugated DTX and polymer 5e, and unconjugated DTX following 2,
4, 24, and 72 h treatment. IC50 determinations are based on triplicate data. All cells were
incubated for a total of 72 h with treatment solutions replaced with media after the
treatment time had elapsed, after which MTS assay was used to determine cellular
metabolic activity. For statistical comparisons and analysis refer to Supporting
Information.
line MCF-7 was performed, to compare the efficacy of the
conjugate against free DTX. The aminated polymer backbone 5
prior to drug conjugation was tested for cytotoxicity at greater
equivalent concentrations (2, 20, and 200 µg ml-1) than any
conjugate treatment (Supporting Information Figure S3). No
measurable inhibition of cellular metabolic activity was
detected from the backbones alone (F(4, 10) = 0.6006, p = 0.67),
so the polymers were considered non-toxic.
A number of treatment times were investigated in vitro to
better model the more complex in vivo environment which has
barriers such as drug half-life, circulation time, and cellular
interactions to overcome. This allowed the investigation of
cellular interactions and/or time-dependent behaviour of the
free DTX and DTX–polymer conjugates to be observed. For
cytotoxicity assessments, cells were incubated for a total of 72 h
from the addition of treatment-containing media before MTS
assays were performed, with shorter treatment times achieved
by washing the cells and adding fresh cell media after the
desired treatment time had elapsed. As expected, a shorter
treatment time correlated with an increased IC50 concentration
(Figure 3a). Cell cycle arrest is known to occur within 24 h in
MCF-7 cells,43 followed by initiation of apoptosis. The tested
conjugate treatments maintained efficacy comparable to DTX
against MCF-7 cells, albeit with higher IC50 values that are
probably attributable to the slow release of DTX from the
polymer (72 h treatment times shown in Figure 3b). As
expected, varying the treatment time affected the IC50 values;
all conjugates showed no significant difference in efficacy from
DTX for 4 h exposure, while at longer times, conjugates gave IC50
values within one order of magnitude of free DTX but
significantly higher in all cases. It should be stressed that all
conjugates were tested at concentrations normalised to DTX
content; therefore, any differences in IC50 values can be
attributed to polymer conjugation and/or release.
Conclusions
In summary, novel synthetic polymer conjugates of DTX of
differing molecular weights were produced, characterised, and
tested in vitro. Conjugates could be prepared with a highly
competitive DTX loading up to 20% w/w. Cleavage of the ester
bond by hydrolysis and subsequent release of DTX was
demonstrated by CPMG 1H NMR spectroscopy and followed by
HPLC in physiologically relevant conditions. Conjugates were
rapidly internalised in MCF-7 cells. All conjugates showed
efficacy within one order of magnitude of free DTX in vitro,
while the macromolecular carrier was non-toxic at
concentrations up to 1000× the IC50 of the drug, making this
system a potential candidate for in vivo study in which its value
as a drug delivery platform may be further assessed. The
Conjugates 8a-c were synthesised with similar DTX loading
but different polymer backbone molecular weight (Mw);
This journal is © The Royal Society of Chemistry 20xx
J. Name., 2013, 00, 1-3 | 5
Please do not adjust margins

Please do not adjust margins
New Journal of Chemistry
Page 6 of 8
ARTICLE
advantage of this system is the tunability of HEMA to GMA
ratios in the copolymer. The reactive groups present allow a Medium α with 10% v/v fetal bovine serum (FBS), 1× GlutaMAX
great deal of flexibility in attaching imaging probes, and 0.15% w/v NaHCO3 in a humidified incubator at 37 °C and
solubilisation enhancers, and targeting ligands to the system. 5% CO2. Cells were seeded in 96-well plates at a density of 5000
Future work may further examine the effects of PEGylation via cells/well in 50 µl of media. Docetaxel and conjugate treatments
side-grafted chains of varying length in order to optimise in media were applied in triplicate at 50 µl per well. After the
solubility, the addition of targeting and therapeutic ligands, designated treatment time had elapsed, media was removed,
Journal Name
1
2
3
4
5
6
7
8
Cell culture. MCF-7 cells were cultured in Minimum Essential
View Article Online
DOI: 10.1039/D0NJ03425H
9
biodistribution, and the potential for tumour targeting in vivo.
cells were washed with PBS (100 µl per well) for 1 min, then the
PBS was removed and prewarmed media was applied (100 µl
per well). Plates were incubated for a total of 72 h from the
beginning of treatment. Assays were performed using warmed
CellTiter 96 AQueous One Solution Cell Proliferation Assay
(MTS) solution at 20 µl per well. Plates were read for
absorbance at 490 nm 3 h after the reagent was applied.
Datasets were normalised and analysed by four-parameter
nonlinear regression with an inhibition model. For confocal
imaging, cells were seeded on glass coverslips and treated for
2 h with RBITC-labelled DTX–polymer conjugates 8b and 8e
(25 µg ml-1) diluted in OptiMEM media, and fixed with 4%
paraformaldehyde. Cells were stained with CytoPainter
Phalloidin-iFluor 488 and Hoechst 34580 in TBST, and mounted
using Fluoromount G (Southern BioTech) on SuperFrost glass
microscope slides.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Materials and Methods
Conjugate preparation. Copolymer backbones
3 were
synthesised by ATRP as previously described using 2-
(Morpholino)ethyl 2-bromoisobutyrate (ME-Br) as the
initiator.36,44 Copolymers were aminated by the action of
aqueous NH3 (30%, 2 ml, 33 mmol) and triethylamine (2 ml, 14
mmol) on 3 (1 g) in dry MeOH (50 ml) at 60 °C for 72 h, after
which the product was dialysed and collected by lyophilisation.
DTX (25 mg, 30.9 µmol) and succinic anhydride (1.23 eq, 3.81
mg, 38.1 µmol) were combined in dry CH2Cl2 (1.5 ml), followed
by the addition of dry pyridine (5 µl). The solution was protected
from light and stirred at room temperature for 72 h. The crude
product was dried under vacuum and purified on silica gel,
washed with hexanes and eluted with ethyl acetate. The ethyl
acetate fractions were collected and dried under vacuum. DTX-
2ʹ-Suc (20 mg, 22 mmol) and SDPP (1.5 eq, 11 mg, 33 mmol)
were then combined with MeCN (1.5 ml) and Et3N
(approximately 5 eq, 15 µl). The flask was protected from light
and stirred for 12 h. The solvent was removed and the product
was dissolved in ethyl acetate and hexanes (2.5:1). The product
was precipitated onto a silica gel column with hexanes, then
washed twice with hexanes, followed by elution with
EtOAc/hexanes (2.5:1). Fractions were dried under vacuum,
triturated with Et2O, and checked by TLC (CHCl3/MeOH 9:1) and
HPLC (MeCN/water 1:1). 1H NMR (DMSO-d6, 600 MHz) δ (ppm):
0.97 (3H, s, H16/H17), 0.98 (3H, s, H16/H17), 1.37 (9H, s, Boc),
1.50 (3H, s, H19), 1.60 (1H, m, H14), 1.64 (1H, m, H6), 1.68 (3H,
s, H18), 1.85 (1H, m, H14), 2.23 (3H, s, OAc), 2.28 (1H, t, H6),
2.78 (m, H2ʺ), 2.80 (s, NHS), 2.98 (2H, t, H3ʺ), 3.63 (m, 3H), 3.89
(1H, m, H20), 4.00 (m, H20), 4.00 (m, H7), 4.45 (1H, s, OH1), 4.9
(1H, d, H5), 4.94 (1H, s, OH10), 5.04 (1H, br, OH7), 5.08 (1H, s,
H10), 5.1 (1H, s, H2ʹ), 5.1 (1H, s, H3ʹ), 5.40 (1H, d, H2), 5.80 (1H,
t, H13), 7.18 (1H, t, para Ar2), 7.36 (2H, d, ortho Ar2), 7.41 (2H,
t, meta Ar2), 7.64 (2H, t, meta Ar1), 7.72 (1H, t, para Ar1), 7.87
(1H, br, NH), 7.98 (2H, d, ortho Ar1). 13C NMR (150 MHz, DMSO-
d6, δ): 9.9 (C19), 13.7 (C18), 22.56 (CH3, OAc), 27.5 (CH3, Boc),
55.1 (C3ʹ), 57.0 (C8), 70.8 (C7), 74.8 (C2), 75.0 (C2ʹ), 78.5 (spiro,
Boc), 83.8 (C5), 127.4 (ortho, Ar2), 128.6 (meta, Ar2), 128.7
(meta, Ar1), 136.8 (tert, Ar2), 165.4 (tert C=O, Ar1); HRMS (ESI,
m/z): [DTX-2ʹ-Suc-NHS + Na]+ calcd for C51H60N2O19Na, 1027.37;
found, 1027.37. DTX–polymer conjugates 8 were synthesised by
combining dry aminated copolymer 5 (29 mg) and DTX-2ʹ-Suc-
NHS (30 mg), and adding DMF (dry, 1.5 ml) and triethylamine
(20 µl). The solution was protected from light and left stirring at
room temperature for 1 h, then washed twice with CH2Cl2 and
diethyl ether. The product was dissolved in DMF, precipitated in
diethyl ether and collected, or dialysed against MeOH/water.
Conflicts of interest
There are no conflicts to declare.
Acknowledgements
The authors acknowledge the facilities, and the scientific and
technical assistance of Microscopy Australia at the Centre for
Microscopy, Characterisation & Analysis, The University of
Western Australia, a facility funded by the University, State and
Commonwealth Governments. The authors thank Amy
Kretzmann and Kelly Irving for support with cell culture. J.A.K.
acknowledges the support of an Australian Postgraduate Award
and Cancer Council WA top-up scholarship. T.D.C. was the
recipient of
a Peter Doherty NHMRC fellowship and
acknowledges the support of the Raine Medical Research
Foundation.
Author Contributions
C
.W.E., K.S.I., and M.N. designed experiments and developed
the concept. S.E., J.A.K., C.W.E. and M.N. synthesised and
characterised the conjugates. S.E. and G.L.N. performed NMR
characterisation of DTX and conjugates. S.E., M.N. and C.A.B.
performed GPC measurements. C.W.E., J.A.K., S.E., R.S., and
T.D.C. performed in vitro toxicity assays and confocal imaging.
K.S.I., T.D.C., and C.W.E. supervised the project. All authors
discussed the results, assisted with analysis and contributed to
writing the manuscript.
References
1
F. Bray, J. Ferlay, I. Soerjomataram, R. L. Siegel, L. A. Torre and A. Jemal, CA: A
Cancer Journal for Clinicians, 2018, 68, 394–424.
6 | J. Name., 2012, 00, 1-3
This journal is © The Royal Society of Chemistry 20xx
Please do not adjust margins

Page 7 of 8
New Journal of Chemistry
Please do not adjust margins
Journal Name
ARTICLE
1
2
3
4
5
6
7
8
9
2
3
4
5
Y. Tang, Y. Wang, M. F. Kiani and B. Wang, Clinical Breast Cancer, 2016, 16, 335–
343.
C. Denkert, C. Liedtke, A. Tutt and G. von Minckwitz, The Lancet, 2017, 389, 2430–
2442.
23 C. W. Evans, M. Fitzgerald, T. D. Clemons, M. J. House, B. S. Padman, J. A. Shaw, M.
View Article Online
Saunders, A. R. Harvey, B. Zdyrko, I. Luzinov, G. A. Silva, S. A. Dunlop and K. S. Iyer,
DOI: 10.1039/D0NJ03425H
ACS Nano, 2011, 5, 8640–8648.
24 D. Peer, J. M. Karp, S. Hong, O. C. Farokhzad, R. Margalit and R. Langer, Nature
Nanotech, 2007, 2, 751–760.
25 V. P. Torchilin, Eur J Pharm Sci, 2000, 11 Suppl 2, S81-91.
26 R. Duncan, Nat. Rev. Cancer, 2006, 6, 688–701.
27 J. Cao, Z. Chen, J. Chi, Y. Sun and Y. Sun, Artificial Cells, Nanomedicine, and
Biotechnology, 2018, 46, 817–830.
28 Y. Yuan, J. Liu and B. Liu, Angew. Chem. Int. Ed., 2014, 53, 7163–7168.
29 M. Kanamala, W. R. Wilson, M. Yang, B. D. Palmer and Z. Wu, Biomaterials, 2016,
85, 152–167.
S. J. Luen, R. Salgado, S. Fox, P. Savas, J. Eng-Wong, E. Clark, A. Kiermaier, S. M.
Swain, J. Baselga, S. Michiels and S. Loi, Lancet Oncol., 2017, 18, 52–62.
S. M. Swain, J. Baselga, S.-B. Kim, J. Ro, V. Semiglazov, M. Campone, E. Ciruelos, J.-
M. Ferrero, A. Schneeweiss, S. Heeson, E. Clark, G. Ross, M. C. Benyunes and J.
Cortés, N Engl J Med, 2015, 372, 724–734.
A. Tutt, H. Tovey, M. C. U. Cheang, S. Kernaghan, L. Kilburn, P. Gazinska, J. Owen, J.
Abraham, S. Barrett, P. Barrett-Lee, R. Brown, S. Chan, M. Dowsett, J. M. Flanagan,
L. Fox, A. Grigoriadis, A. Gutin, C. Harper-Wynne, M. Q. Hatton, K. A. Hoadley, J.
Parikh, P. Parker, C. M. Perou, R. Roylance, V. Shah, A. Shaw, I. E. Smith, K. M.
Timms, A. M. Wardley, G. Wilson, C. Gillett, J. S. Lanchbury, A. Ashworth, N.
Rahman, M. Harries, P. Ellis, S. E. Pinder and J. M. Bliss, Nat. Med., 2018, 24, 628–
637.
6
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
30 H. Maeda, J Drug Target, 2017, 25, 781–785.
31 H. Maeda, Advanced Drug Delivery Reviews, 2015, 91, 3–6.
32 P. Zhang, H. Zhang, W. He, D. Zhao, A. Song and Y. Luan, Biomacromolecules, 2016,
17, 1621–1632.
33 P. Ha-Lien Tran, T. Wang, C. Yang, T. T. D. Tran and W. Duan, Mater Sci Eng C
Mater Biol Appl, 2020, 107, 110346.
34 Z. H. Mirzaie, S. Irani, R. Mirfakhraie, S. M. Atyabi, M. Dinarvand, R. Dinarvand, R.
Varshochian and F. Atyabi, Chem Biol Drug Des, 2016, 88, 850–858.
35 Y. Guo, P. Zhang, Q. Zhao, K. Wang and Y. Luan, Macromolecular Bioscience, 2016,
16, 420–431.
36 J. A. Kretzmann, D. Ho, C. W. Evans, J. H. C. Plani-Lam, B. Garcia-Bloj, A. E.
Mohamed, M. L. O’Mara, E. Ford, D. E. K. Tan, R. Lister, P. Blancafort, M. Norret
and K. S. Iyer, Chem Sci, 2017, 8, 2923–2930.
37 J. A. Kretzmann, C. W. Evans, C. Moses, A. Sorolla, A. L. Kretzmann, E. Wang, D. Ho,
M. J. Hackett, B. F. Dessauvagie, N. M. Smith, A. D. Redfern, C. Waryah, M. Norret,
K. S. Iyer and P. Blancafort, Chem. Sci., 2019, 10, 7718–7727.
38 P. S. R. Naidu, N. Gavriel, C. G. G. Gray, C. A. Bartlett, L. M. Toomey, J. A.
Kretzmann, D. Patalwala, T. McGonigle, E. Denham, C. Hee, D. Ho, N. L. Taylor, M.
Norret, N. M. Smith, S. A. Dunlop, K. S. Iyer and M. Fitzgerald, ACS Appl. Mater.
Interfaces, 2019, 11, 22085–22095.
39 M. Skwarczynski, Y. Hayashi and Y. Kiso, J. Med. Chem., 2006, 49, 7253–7269.
40 A. J. Brandolini and D. D. Hills, NMR spectra of polymers and polymer additives,
Marcel Dekker, New York, 2000.
41 J. Fisher and A. D. Bain, Modern NMR techniques for synthetic chemistry, CRC
Press, Boca Raton, FL, 2015.
42 M. F. Attia, N. Anton, J. Wallyn, Z. Omran and T. F. Vandamme, J. Pharm.
Pharmacol., 2019, 71, 1185–1198.
7
8
9
H. Gelderblom, J. Verweij, K. Nooter and A. Sparreboom, Eur. J. Cancer, 2001, 37,
1590–1598.
C. D. M. Churchill, M. Klobukowski and J. A. Tuszynski, Chem Biol Drug Des, 2015,
86, 1253–1266.
B. A. Weaver, Mol. Biol. Cell, 2014, 25, 2677–2681.
10 L. Eckhoff, A. Knoop, M. B. Jensen and M. Ewertz, Eur. J. Cancer, 2015, 51, 292–
300.
11 R. C. Wang, X. Chen, A. M. Parissenti, A. A. Joy, J. Tuszynski, D. N. Brindley and Z.
Wang, PLoS ONE, 2017, 12, e0182400.
12 E. Miele, G. P. Spinelli, E. Miele, F. Tomao and S. Tomao, Int J Nanomedicine, 2009,
4, 99–105.
13 J. A. Yared and K. H. R. Tkaczuk, Drug Des Devel Ther, 2012, 6, 371–384.
14 N. P. Desai, V. Trieu, L. Y. Hwang, R. Wu, P. Soon-Shiong and W. J. Gradishar,
Anticancer Drugs, 2008, 19, 899–909.
15 N. Goodarzi, M. H. Ghahremani, M. Amini, F. Atyabi, S. N. Ostad, N. Shabani Ravari,
N. Nateghian and R. Dinarvand, Chem Biol Drug Des, 2014, 83, 741–752.
16 Y. Luo and G. D. Prestwich, Bioconjugate Chem., 1999, 10, 755–763.
17 F. Seidi, R. Jenjob and D. Crespy, Chem. Rev., 2018, 118, 3965–4036.
18 L. Tang, T. M. Fan, L. B. Borst and J. Cheng, ACS Nano, 2012, 6, 3954–3966.
19 D. Rosenblum, N. Joshi, W. Tao, J. M. Karp and D. Peer, Nat Commun, 2018, 9,
1410.
20 A. Sorolla, D. Ho, E. Wang, C. W. Evans, C. F. G. Ormonde, R. Rashwan, R. Singh, K.
S. Iyer and P. Blancafort, Nanoscale, 2016, 8, 9343–9353.
21 A. Sorolla, E. Wang, T. D. Clemons, C. W. Evans, J. H. Plani-Lam, E. Golden, B.
Dessauvagie, A. D. Redfern, K. Swaminathan-Iyer and P. Blancafort, Nanomedicine:
Nanotechnology, Biology and Medicine, 2019, 20, 102003.
22 N. T. Huynh, E. Roger, N. Lautram, J.-P. Benoît and C. Passirani, Nanomedicine,
2010, 5, 1415–1433.
43 F. Esmaeili, R. Dinarvand, M. H. Ghahremani, M. Amini, H. Rouhani, N. Sepehri, S.
N. Ostad and F. Atyabi, J Pharm Sci, 2009, 98, 2718–2730.
44 D. L. Morse, H. Gray, C. M. Payne and R. J. Gillies, Mol. Cancer Ther., 2005, 4, 1495–
1504.
45 J. V. M. Weaver, I. Bannister, K. L. Robinson, X. Bories-Azeau, S. P. Armes, M.
Smallridge and P. McKenna, Macromolecules, 2004, 37, 2395–2403.
This journal is © The Royal Society of Chemistry 20xx
J. Name., 2013, 00, 1-3 | 7
Please do not adjust margins

New Journal of Chemistry
Page 8 of 8
View Article Online
DOI: 10.1039/D0NJ03425H
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60