Mucus-responsive functionalized emulsions: Design, synthesis and study of novel branched polymers as functional emulsifiers

Article abstract of DOI:10.1039/d0ra05820c

Mucus lines the moist cavities throughout the body, acting as barrier by protecting the underlying cells against the external environment, but it also hinders the permeation of drugs and drug delivery systems. As the rate of diffusion is low, the development of a system which could increase retention time at the mucosal surface would prove beneficial. Here, we have designed a range of branched copolymers to act as functional mucus-responsive oil-in-water emulsifiers comprising the hydrophilic monomer oligo(ethylene glycol) methacrylate and a hydrophobic dodecyl initiator. The study aimed to investigate the importance of chain end functionality on successful emulsion formation, by systematically replacing a fraction of the hydrophobic chain ends with a secondary poly(ethylene glycol) based hydrophilic initiator in a mixed-initiation strategy; a decrease of up to 75 mole percent of hydrophobic chain ends within the branched polymer emulsifiers was shown to maintain comparative emulsion stability. These redundant chain ends allowed for functionality to be incorporated into the polymers via a xanthate based initiator containing a masked thiol group; thiol groups are known to have mucoadhesive character, due to their ability to form disulfide bonds with the cysteine rich areas of mucus. The mucoadhesive nature of emulsions stabilised by thiol-containing branched copolymers was compared to non-functional emulsions in the presence of a biosimilar mucosal substrate and enhanced adherence to the mucosal surface was observed. Importantly, droplet rupture and mucus triggered release of dye-containing oil was seen from previously highly-stable thiol-functional emulsions; this observation was not mirrored by non-functional emulsions where droplet integrity was maintained even in the presence of mucus.

Full text of DOI:10.1039/d0ra05820c

RSC Advances
View Article Online
View Journal | View Issue
PAPER
Mucus-responsive functionalized emulsions:
design, synthesis and study of novel branched
Cite this: RSC Adv., 2020, 10, 30463
polymers as functional emulsifiers†
ab
ab
a
ab
Stephanie E. Edwards, Sean Flynn, James J. Hobson, Pierre Chambon,
a
ab
Helen Cauldbeck and Steve P. Rannard
*
Mucus lines the moist cavities throughout the body, acting as barrier by protecting the underlying cells
against the external environment, but it also hinders the permeation of drugs and drug delivery systems.
As the rate of diffusion is low, the development of a system which could increase retention time at the
mucosal surface would prove beneficial. Here, we have designed a range of branched copolymers to act
as functional mucus-responsive oil-in-water emulsifiers comprising the hydrophilic monomer
oligo(ethylene glycol) methacrylate and a hydrophobic dodecyl initiator. The study aimed to investigate
the importance of chain end functionality on successful emulsion formation, by systematically replacing
a fraction of the hydrophobic chain ends with a secondary poly(ethylene glycol) based hydrophilic
initiator in a mixed-initiation strategy; a decrease of up to 75 mole percent of hydrophobic chain ends
within the branched polymer emulsifiers was shown to maintain comparative emulsion stability. These
redundant chain ends allowed for functionality to be incorporated into the polymers via a xanthate
based initiator containing a masked thiol group; thiol groups are known to have mucoadhesive
character, due to their ability to form disulfide bonds with the cysteine rich areas of mucus. The
mucoadhesive nature of emulsions stabilised by thiol-containing branched copolymers was compared to
non-functional emulsions in the presence of a biosimilar mucosal substrate and enhanced adherence to
the mucosal surface was observed. Importantly, droplet rupture and mucus triggered release of dye-
containing oil was seen from previously highly-stable thiol-functional emulsions; this observation was
not mirrored by non-functional emulsions where droplet integrity was maintained even in the presence
of mucus.
Received 3rd July 2020
Accepted 22nd July 2020
DOI: 10.1039/d0ra05820c
rsc.li/rsc-advances
material and prevent infection; in addition, the permeation of
many pharmaceutical agents is also restricted in this way.
Introduction
Mucous membranes create the moist exterior of various regions Mucous membranes have, therefore, been a focus for drug
of the body including oral, gastrointestinal (GI), genital and delivery over many years and mucoadhesion and mucus
7
ocular surfaces. For example, mucus is secreted by goblet cells permeability have been the subject of much research.
directly onto epithelium cells within the GI tract, forming
1
Structurally, mucus comprises a water-swollen network of
a mobile outer layer, that is relatively quickly replaced, and gel-forming mucins which are cysteine-rich high molecular
2
a stationary gel–like layer, that is adhered to the surface of the weight glycosylated proteins. Disulde bridges between cysteine
goblet cells. Mucosa varies across different anatomical sites residues contribute to the physical properties of the mucosal
with different life-cycles and thicknesses that depend primarily membrane; these sulfur-containing domains offer a chemical
3
–5
on its specic local function.
As a biologically-derived target for mucoadhesion and mucopermeation strategies.
6
hydrogel with dened viscoelasticity, rheology and mesh size, Mucoadhesion is generally regarded as progressing through
mucous membranes act to moderate the passage of exogenous a two-step process, namely contact and consolidation. In situ-
8
ations where adhesion to mucus occurs within a predominantly
liquid environment, the contact stage may be considered using
Department of Chemistry, University of Liverpool, Crown Street, L69 7ZD, UK. E-mail: conventional DLVO theory, as the resultant contradictory forces
a
srannard@liv.ac.uk
b
of attraction and repulsion must favour a physical interaction;
a positive wetting interaction will also favour the contact stage.
The material in contact with the mucus may be removed
through various shear or stress modes and therefore consoli-
dation of the interaction is required to generate a long-acting
Materials Innovation Factory, University of Liverpool, Oxford Street, Liverpool L7
3
NY, UK
Electronic supplementary information (ESI) available: H and C NMR spectra,
1
13
†
SEC chromatograms, surface tension measurements, dynamic light scattering
data and emulsion characterisation. See DOI: 10.1039/d0ra05820c
This journal is © The Royal Society of Chemistry 2020
RSC Adv., 2020, 10, 30463–30475 | 30463

View Article Online
RSC Advances
Paper
adhesion. In a mainly aqueous medium, this may be achieved weight branched copolymers interact with oil droplet surfaces
9
in several ways including entrapment, non-covalent intermo- through multiple attachment sites, providing a contrast to
lecular forces (e.g. electrostatic, van der Waals' and hydrogen linear polymer surfactants which suffer from single dynamic
bonding interactions), and the formation of covalent bonds interactions, and making them more akin to Pickering emul-
through chemical reactions (e.g. disulde formation and sions, Fig. 1C, as evidenced by their remarkable stability and
24
Michael addition).
resistance to dilution.
Thiol-functional materials and thiol-bearing polymers, oen
Herein, we aimed to investigate the impact of decreasing the
10–12
called “thiomers”,
have been studied specically to interact number of hydrophobic chain-ends within the branched
with cysteine residues within mucins, forming disulde bonds copolymer stabiliser as a strategy for introducing thiol func-
either through thiol/disulde exchange or thiol–thiol reactions tionality to emulsion droplets and controllably inducing
13
within the mucus network; evidence for these mechanisms has mucoadhesion. Within this model study we monitored emul-
1
4–17
been established in several reports.
thiol-functional polymers have been used to produce tablets,
polymeric micelles
To enable drug delivery, sion storage stability over prolonged periods and the triggering
17
of demulsication on contact with biosimilar mucus surfaces.
and microparticles. Recently, alkyl- To the best of our knowledge, this represents the rst demon-
18,19
20
modied carbomers (lightly crosslinked poly(acrylic acid)) were stration of a triggered mucus-responsive emulsion system
modied with 4-aminothiophenol, L-cysteine or D/L-homocysteine enabled by novel branched copolymer emulsiers.
to form a novel class of emulsiers with the aim of generating
21
mucoadhesive emulsions; the emulsication of medium chain
triglycerides was reported with varying stabilities, especially if the
thiomers used were able to oxidise or crosslink prior to emulsi-
Results and discussion
Investigating chain-end criticality for branched copolymer
emulsiers
cation. These emulsions were also shown to adhere to porcine
buccal mucosa, hence demonstrating the concept of mucoad-
hesive oil-in-water (O/W) emulsions.
Polymerisation studies and copolymer characterisation. The
synthesis of branched vinyl copolymers using the incorporation
In recent work, we have shown that branched vinyl polymers of low concentrations of divinyl monomers (less than 1 per
made by conventional and controlled radical polymerisation are propagating primary chain) has been the subject of several
22–25
26
able to act is highly efficient emulsiers.
Branching was research reports. The mixing of differing monovinyl monomer
achieved by the copolymerisation of the macromonomer oli- chemistries within the primary chains of the complex polymer
2
7,28
go(ethylene glycol methacrylate), (OEGMA) with the divinyl architectures
has also been used to introduce main-chain
however, the joining together of polymer
2
2,23
monomer ethylene glycol dimethacrylate (EGDMA), leading to functionality;
a large number of hydrophilic primary polymer chains bridged chains via this approach opens a unique opportunity to also
by a small number of branch points. Through the utilization of control chain-end functionality, as demonstrated by our group
either hydrophobic initiators (controlled radical polymerisa- in the formation of hyperbranched-polydendrons with varying
tion) or hydrophobic chain transfer agents (conventional combinations of dendritic and polyethylene glycol (PEG)
29–31
radical synthesis), amphiphilicity can be introduced into the chains.
As mentioned above, we have recently reported the
hydrophilic water-soluble branched polymer at the end of each formation and application of branched copolymers consisting
ꢀ
1
conjoined primary chain, Fig. 1B. The resulting high molecular of OEGMA (M ¼ 300 g mol ) and EGDMA, initiated by dodecyl
n
a-bromoisobutyrate, 1 (Dod-Br, Scheme 1), under atom transfer
radical polymerisation (ATRP) conditions, as highly efficient
polymeric surfactants; the copolymers contain a hydrophobic
24
dodecyl group at every chain-end.
We hypothesised that initiating this copolymerisation with
mixed initiator feedstocks would allow the creation of branched
copolymers with varying chain-end composition; however, the
decrease in hydrophobic chain-ends may potentially impair the
surfactant behaviour of the branched copolymers. To study this
in detail, a series of branched copolymers with systematically
varying numbers of hydrophilic and hydrophobic chain-ends
was synthesised to establish the critical content of hydro-
phobic (lipophilic) chain-ends within the branched copolymer
structures required for stable O/W emulsion formation.
As seen in Scheme 1A, co-initiation of the linear polymeri-
sation of OEGMA with varying ratios of dodecyl-derived, 1, and
PEG17-derived, 2 (PEG(17)-Br; starting material PEG-mono
ꢀ1
methyl ether 750 g mol ), initiators leads to a mixed linear
polymer product where a fraction of the polymer chains possess
hydrophilic PEG chain-ends ([PEG(17)1.00]–p(OEGMA50)), and
hence are exclusively hydrophilic, whilst the remaining fraction
Fig. 1 Schematic representation of an oil-in-water emulsion droplet
stabilized by: (A) conventional amphiphilic linear copolymer stabilisers;
(
B) branched copolymer stabiliser with multiple attachment through
hydrophobic chain-ends; and (C) particle adsorption.
30464 | RSC Adv., 2020, 10, 30463–30475
This journal is © The Royal Society of Chemistry 2020

View Article Online
Paper
RSC Advances
utilising the redundancy to introduce functional groups may
become available.
A series of branched OEGMA/EGDMA copolymers were,
therefore, synthesised via a mixed initiator, copper-catalysed
ATRP strategy in an isopropyl alcohol (IPA)/water mixture
ꢁ
(
92.5/7.5 v/v%) at 40 C. All coinitiated copolymerisations tar-
geted a number average degree of polymerisation (DP
monomer units ([M] /S[I]
primary chains within the various targeted branched structures.
The mixed initiator ratios used systematically varying 1 : 2
molar ratios of 1.00 : 0.00, 0.90 : 0.10, 0.75 : 0.25, 0.50 : 0.50,
n
) of 50
0
0
¼ 50) to ensure consistency for the
0.25 : 0.75, 0.10 : 0.90 and 0.00 : 1.00; a consistent EGDMA : S
[
I] molar ratio of 0.80 : 1 was also employed ([B] /S[I]
0
0
0
¼ 0.80)
as this ratio allows a signicant degree of branching whilst
avoiding gelation. The linear polymers [PEG(17)1.00]–
p(OEGMA50) and [Dod1.00]–p(OEGMA50) were also synthesised,
to allow comparison of synthesis conditions for each initiator
and emulsication performance of mixed polymer samples as
a control. Branched copolymerisations oen require extended
1
reaction times, therefore each reaction was monitored by H
nuclear magnetic resonance (NMR) spectroscopy and termi-
nated when high conversion ($99%) was established. Triple
detection size exclusion chromatography (TD-SEC), using
a dimethylformamide (DMF)/0.01 M LiBr eluent, was utilised to
n
determine number average molecular weight (M ), weight
average molecular weight (M ) and dispersity (Đ), Table 1.
w
The M values of the linear polymer samples were higher than
n
targeted, with Đ values indicating non-ideal initiator efficiencies
and the inherent dispersity of the PEG17-derived macroinitiator.
The branched copolymerisations showed relatively consistent M
n
values, in general, suggesting only a minor difference in the
behaviour of the two initiators and comparable initiator effi-
ciencies during the early stages of copolymerisation.
As expected, the M and Mw values were consistently higher
n
for the branched copolymers than their linear analogues with
correspondingly higher dispersities (ESI Fig. S7–S9†). Within
these broad distributions, a weight fraction analysis of the TD-
SEC data indicated that between approximately 6–34 wt% of
ꢀ1
Scheme 1 Co-initiation of alcoholic atom transfer radical copoly-
merisation of oligo(ethylene glycol methacrylate) and ethylene glycol
dimethacrylate to form: (A) a mixed linear polymer sample containing
the various samples have molecular weights >500 kg mol
therefore containing > 10 conjoined chains), ESI Fig. S10, S11
(
and Table S1.† As such, although there will be heterogeneity of
the mixed initiator-derived chain-ends, a signicant proportion
of branched copolymers will possess mixtures of PEG and Dod
chain-ends even at the extremes of the 1 : 2 ratios targeted, Fig. 2.
hydrophilic [PEG(17)1.00]–p(OEGMA50) and amphiphilic [Dod1.00]–
p(OEGMA50) chains, and (B) the branched copolymer with varying
ratios of PEG17 and dodecyl chain ends.
will carry
a hydrophobic dodecyl chain-end ([Dod1.00]–
p(OEGMA )), and possess amphiphilic character. Conversely, Aqueous solution behaviour and emulsication studies
5
0
the introduction of a low molar concentration of EGDMA,
Scheme 1B, will combine the mixed linear population into
a range of larger structures with an overall controlled average of
PEG17 and dodecyl chain-ends across the full polymer distri-
bution. If a substantial number of hydrophobic dodecyl chain-
ends could be replaced with hydrophilic PEG17 chain-ends,
whilst maintaining emulsier properties, an indication of
chain-end redundancy would be derived and the potential for
All branched copolymers showed high aqueous solubility
>5 wt%) and no observable solution differences across the
samples with varying overall chain-end composition; lower
critical solution temperature (LCST) studies exhibited a trend to
lower values with increasing hydrophobic chain-end content,
(
and branched [PEG1.00]–p(OEGMA -co-EGDMA0.80) showed
50
slightly lower values than its linear analogue; a relatively narrow
range of transition temperatures was observed across the series
ꢁ
(
approximately 61–67 C), ESI, Fig S12.†
This journal is © The Royal Society of Chemistry 2020
RSC Adv., 2020, 10, 30463–30475 | 30465

View Article Online
RSC Advances
Paper
Table 1 Triple detection size exclusion chromatography analysis of OEGMA-based linear homopolymers and branched copolymers initiated
using varied molar ratios of Dod-Br and PEG17-Br
a
n
ꢀ1
a
w
ꢀ1
a
Architecture
Copolymer
M
(g mol
)
M
(g mol
)
Đ
Linear homopolymers
Branched copolymers
[Dod1.00]–p(OEGMA50
)
30 750
38 000
57 600
22 300
62 500
54 500
76 400
40 050
63 600
39 750
70 350
1.29
1.85
4.09
6.10
4.12
3.00
5.37
7.59
2.66
[PEG(17)1.00]–p(OEGMA50
)
[Dod1.00]–p(OEGMA50-co-EGDMA0.80
)
235 700
136 100
257 700
163 700
410 600
303 800
169 800
[
[
[
[
[
[
Dod0.90/PEG(17)0.10]–p(OEGMA50-co-EGDMA0.80
)
)
)
)
)
Dod0.75/PEG(17)0.25]–p(OEGMA50-co-EGDMA0.80
Dod0.50/PEG(17)0.50]–p(OEGMA50-co-EGDMA0.80
Dod0.25/PEG(17)0.75]–p(OEGMA50-co-EGDMA0.80
Dod0.10/PEG(17)0.90]–p(OEGMA50-co-EGDMA0.80
PEG(17)1.00]–p(OEGMA50-co-EGDMA0.80
)
a
1
TD-SEC using DMF/0.01 M LiBr eluent; all polymerisation achieved $99% monomer conversion as determined by H NMR (CDCl
3
,400 MHz).
The varying branched copolymer structures, Fig. 2, were also contact angle of the various solutions to range from approxi-
ꢁ
studied using static contact angle measurement of aqueous mately 99–105 ; the clear inference being that redundant
branched copolymer surfactant solutions (5 wt%). Aqueous hydrophobic chain-ends are present at the droplet surface for
solutions were placed onto a hydrophobic polytetrauoro- solutions containing [Dod1.00]–p(OEGMA50-co-EGDMA0.80) and
ethylene substrate to evaluate the presence and impact of the these are somewhat removed and replaced by the more hydro-
varying hydrophilic/hydrophobic chain-ends (Fig. 3A(i)–(iv)). All philic PEG17 chain-ends in the solutions of copolymers con-
solutions containing branched copolymers showed a decrease taining mixed chain-end functionality. The contribution of the
ꢁ
ꢁ
in contact angle from deionised water (125 ꢂ 3.6 , Fig. 3A(i)), hydrophobic polymethacrylate backbone to the changes in
indicating the presence of the branched copolymer at the air/ observed contact angles must also not be ignored; however, this
water interface and an increase in hydrophobicity (ESI, Table remains consistent within the polymer structures and may
S2†). The lowest contact angle was observed for [Dod1.00]– become more prominent with the changing chain-end
ꢁ
ꢁ
p(OEGMA50-co-EGDMA0.80) solutions (80 ꢂ 2.2 ) and the chemistry.
introduction of PEG(17)-derived chain-ends increased the Surface tension measurements were conducted to study the
impact of the increasing number of hydrophilic PEG-derived
chain-ends within the branched copolymer structures (ESI,
Table S3, Fig. S13†). Interestingly, changes to copolymer end-
group functionality across all materials had little impact on
Fig. 3 Assessment of branched copolymer aqueous solution behav-
iours. (A) The relationship between branched copolymer end-group
composition and contact angle observed for droplets of aqueous
copolymer solutions on a hydrophobic PTFE substrate, where (i) pure
water, (ii) [PEG(17)]–p(OEGMA50-co-EGDMA0.80), (iii) [Dod0.75/PEG(17)
0
.25]–p(OEGMA50-co-EGDMA0.80) and (iv) [Dod1.00]–p(OEGMA50-co-
EGDMA0.80). (B) Overlaid plots obtained from surface tensiometry
measurements of aqueous linear homopolymer solutions of
Fig. 2 Schematic representation of branched copolymer structures [Dod1.00]–p(OEGMA50) (black circles) and [PEG(17)1.00]–p(OEGMA50
)
with varying hydrophobic/hydrophilic chain-end compositions. (red circles).
30466 | RSC Adv., 2020, 10, 30463–30475
This journal is © The Royal Society of Chemistry 2020

View Article Online
Paper
RSC Advances
their CMC behaviour. The intercept of the rst plateau of the
surface tension curves were observed at concentrations of
ꢀ
8
ꢀ8
ꢀ1
between 4.72 ꢃ 10 (ꢂ8%) and 7.40 ꢃ 10 (ꢂ2%) mg L for
linear polymers [Dod ]–p(OEGMA ) and PEG(17)1.00–
1
.00
50
p(OEGMA50) respectively (Fig. 3B), suggesting a role for the
hydrophobic methacrylate backbone in adsorption to the oil
droplet surface. Whilst the slightly lower CMC obtained for
[Dod1.00]–p(OEGMA50) could be attributed to the presence of the
hydrophobic dodecyl chain-ends, comparable CMC values likely
arise due to the low weight fraction of the polymer chains-ends
(<2 wt% in all cases based on Mn(GPC)).
This is supported by the consistent CMC values obtained for
branched copolymers containing mixed chain-end functionality
ESI Fig. S14†). All branched copolymer surfactants showed
(
complex surface tension behaviour with increasing copolymer
concentration; initial plateaus were followed by subsequent
further decreases in surface tension at higher concentration.
This behaviour is well reported for complex mixtures such as
polymeric/small molecule surfactant combinations, and there-
fore is readily rationalised as being derived from the distribu-
tion of species within the statistically branched copolymer
molecular weight distribution (ranging from linear polymers
through to highly branched structures). CMC values obtained
from branched copolymers containing mixed chain-end func-
ꢀ
8
ꢀ8
tionality ranged from 1.08 ꢃ 10
(ꢂ16%) to 4.74 ꢃ 10
ꢀ
1
(
ꢂ2%) mg L and showed no clear trend between chain-end
composition and CMC values. This data indicates that,
despite their contrasting chain-end compositions, branched
copolymer emulsiers are expected to adopt similar confor-
mations in aqueous solution and would exhibit similar
responses to changes in concentration. Furthermore, this
demonstrates that it would be possible to manipulate branched
copolymer emulsifying properties with minimal impact on their
aqueous solution behaviour.
Evaluation of the branched copolymers as emulsiers (5 mg
ꢀ
1
mL ) was conducted using high shear homogenisation of
dodecane (1 : 1 v/v ratio) to create O/W emulsions, Fig. 4.
Dodecane was selected as the disperse phase of the O/W emul-
sions due to its similarity to the dodecyl hydrophobic chain-end, Fig. 4 Analysis of O/W emulsions stabilised by branched copolymer
emulsifiers with mixed chain-end functionality. (A) Digital photographs
taken after 3 years storage at ambient temperature of dodecane-in-
water emulsions stabilised with: (i) [Dod1.00]–p(OEGMA50-co-
EGDMA0.80), (ii) [Dod0.75/PEG(17)0.25]–p(OEGMA50-co-EGDMA0.80), (iii)
introduced by the Dod-Br initiator, and the favourable interac-
tions that should therefore occur with the resultant oil droplets.
In the absence of a branched copolymer emulsier, stable
emulsions could not be produced, with rapid demulsication [Dod0.50/PEG(17)0.50]–p(OEGMA50-co-EGDMA0.80),
and resulting phase separation within approximately two PEG(17)0.75]–p(OEGMA50-co-EGDMA0.80 and
(iv)
[Dod0.25/
)
(v)
[PEG(17)1.00]–
p(OEGMA50-co-EGDMA0.80). (B) Study of emulsion droplet stability
over 40 days (laser diffraction) and (C) optical microscopy images of
dodecane emulsions after 11 months storage at ambient temperature;
minutes of standing; similarly, all samples emulsied with linear
polymers, or mixtures of linear polymers with varying
hydrophilic/hydrophobic ratios of chain ends, demulsied rela-
(i) [Dod1.00]–p(OEGMA50-co-EGDMA0.80), (ii) [Dod0.75/PEG(17)0.25]–
tively rapidly on standing for less than one week (ESI Fig. S15†). p(OEGMA50-co-EGDMA0.80), (iii) [Dod0.50/PEG(17)0.50]–p(OEGMA50
For all samples containing branched copolymer surfactants co-EGDMA0.80), (iv)
EGDMA0.80), 20ꢃ magnification, scale bar ¼ 100 mm.
-
[Dod0.25/PEG(17)0.75]–p(OEGMA50-co-
with various ratios of Dod and PEG(17) chain-ends, stable
emulsions were formed. Creaming of the O/W emulsion was
observed aer 24 hours equilibration due to density differences;
however, no oil separation was apparent with emulsions char-
acteristically white and opaque and stable for >3 years at
ambient temperature (Fig. 4A(i)–(iv)). Quantitative analysis by
laser diffraction spectroscopy (ESI, Table S4, Fig. S16†) showed
ranging from 11.9–15.3 mm with a trend towards larger droplet
diameters with decreasing Dod content within the branched
copolymer solutions of identical concentration. Interestingly,
the size distribution of O/W emulsion droplets stabilised by
branched copolymer emulsiers resembled the smaller droplets
highly comparable volume average mean diameters (D[4,3]
)
This journal is © The Royal Society of Chemistry 2020
RSC Adv., 2020, 10, 30463–30475 | 30467

View Article Online
RSC Advances
Paper
within the bimodal size distribution generated by the equiva-
lent mixed linear homopolymer system, described above (ESI
Fig. S17†).
The reduction of the number of hydrophobic chain-ends
appears to be analogous to a reduction in small-molecule
surfactant concentration, therefore a lower surface area may
be stabilised and a smaller number of larger droplets result. As
mentioned above, the dispersity of the co-initiated branched
copolymer samples does also lead to the potential presence of
a fraction of linear [PEG(17)1.00]–p(OEGMA ) and some lightly
50
branched materials having no Dod chain-ends. As may have
been predicted, emulsions prepared using [PEG(17)1.00]–
p(OEGMA50-co-EGDMA0.80) showed no long-term stability, with
complete and rapid demulsication occurring within 72 hours
(ESI Fig. S18†). The role of the hydrophobic backbone in
generating some stability to the emulsions cannot be ruled out;
however, this material emphasises the critical importance of
chain-end chemistry on the formation of stable emulsions.
Observation of the D[4,3] values over 40 days under ambient
conditions for the emulsions stabilised with Dod-containing
branched copolymers revealed very small changes in emulsion
droplet sizes overall (Fig. 4B). The obtained D[4,3] values corre-
lated well with those obtained by optical microscopy imaging
(Fig. 4C(i)–(iv)), which show well-dened spherical droplets with
no signs of coalescence across varying branched copolymer
compositions, even aer several months of storage under
ambient conditions. Laser diffraction was also used to assess
long-term stability of the different O/W emulsions and minimal
changes in the observed distributions and measured D[4,3]
values were seen between samples studied 24 hours aer
emulsication and aer storage under ambient conditions for 3
years (ESI, Fig. S19, Table S5†). Dilution of the Dod-containing
branched copolymer stabilised emulsions from the high
concentration creamed layer through to 6.25% (v/v) also
conrmed the unusual stability of the emulsions (Fig. 5) as re-
ported previously for branched copolymer-stabilised O/W
Fig. 5 Dilution stability of a [Dod1.00]–p(OEGMA50-co-EGDMA0.80)-
stabilised dodecane-in-water emulsion. (A) Optical microscopy
images at dilution factors of; (i) 2-fold, (ii) 4-fold, (iii) 6-fold, and (iv) 32-
fold. (B) Measurement of droplet diameters following dilution in
2
deionised H O, as determined by optical microscopy (ꢂstd. deviation).
24
nanoemulsions. This behaviour is remarkable given the
probable presence of linear and low molecular weight species
within the distribution bearing no hydrophobic chain-ends.
formation of linear polymers under identical conditions to
those used above but initiated with 3, to yield [Xan1.00]–
p(OEGMA50), was investigated and shown to be comparable to
the synthesis of [Dod1.00]–p(OEGMA50
) and [PEG(17)1.00]–
p(OEGMA ). The initiator efficiency of 3 does appear to be
5
0
Thiol functional to branched copolymer emulsiers
slightly higher than the other initiators (approximately 67% for
; approximately 50% for 1) suggesting a slightly higher number
The ability to introduce redundant chain-ends into the
branched copolymer emulsiers, whilst maintaining efficient
emulsication, offers the potential to add functional groups to
the termini of a controlled number of primary chains across the
polymer distribution without compromising performance of
the emulsiers. Here, we have targeted thiol functionality, to
facilitate mucus adhesion, through the use of a mixed initiation
using 1 and a previously reported xanthate-functional ATRP
initiator (Xan-Br), 3, Scheme 2.
3
of actual xanthate-bearing chains would be present in branched
copolymer samples using the initiator than targeted through
the nominal initiator molar ratios. The formation of a series of
comparable branched copolymerisations utilising EGDMA and
mixed 1 : 3 coinitiation achieved $99% monomer conversion in
all cases and samples were analysed by TD-SEC to determine
molecular weights and dispersities, Table 2 (ESI, Fig. S20 and
S21†).
To compare the xanthate-functional branched copolymers
with those containing mixed Dod/PEG17 chain-ends, the TD-
SEC data was analysed to provide a weight fraction analysis,
indicating that, again, between approximately 6–19 wt% of the
species in branched copolymer samples have molecular weights
As previously reported for analogous materials, the resulting
y x
[Xan /Dod ]–p(OEGMA50-co-EGDMA0.80) branched copolymers,
with varying ratios of Dod and xanthate chain ends, may be
readily deprotected using n-butylamine to liberate the thiol
functionality and create a series of functional emulsiers
y x
denoted as [SH /Dod ]–p(OEGMA50-co-EGDMA0.80). The
30468 | RSC Adv., 2020, 10, 30463–30475
This journal is © The Royal Society of Chemistry 2020

View Article Online
Paper
RSC Advances
p(OEGMA50-co-EGDMA0.80),
[SH0.75/Dod0.25]–p(OEGMA50-co-
EGDMA0.80) and [SH0.95/Dod0.05]–p(OEGMA -co-EGDMA0.80).
50
1
H NMR analysis was used to conrm the removal of xanthate
groups from branched copolymer chain-ends post deprotection
evident by the disappearance of the chemical shi attributed to
the methylene protons of the xanthate group at 4.66 ppm (ESI,
Fig. S23†).
Aqueous solutions of these materials were studied using
surface tensiometry, and similar complex behaviour was
observed (ESI, Fig. S24†), with the rst plateau regions observed
ꢀ
6
ꢀ6
ꢀ5
at 9.12 ꢃ 10 (ꢂ8.7%), 9.13 ꢃ 10 (ꢂ8.4%), 1.55 ꢃ 10
ꢀ
5
ꢀ1
(
ꢂ16.2%) and 1.83 ꢃ 10 (ꢂ2.0%) mg mL respectively (ESI,
Table S7†). This correlation of surface tension with thiol
content was more marked than the previous Dod/PEG materials
and the lack of the additional PEG chain-ends generated values
more consistent with those seen when studying [Dod1.00]–
p(OEGMA50-co-EGDMA0.80); this is probably due to the hydro-
phobic nature of the xanthate-bearing chain-ends. Contact
angle measurements were also within the same range as those
seen for Dod/PEG branched copolymers containing varied
ꢁ
chain-end compositions (93–108 , ESI, Fig. S25 and Table S8†)
and efficient emulsication was, therefore, expected.
Emulsication and mucus-responsive studies using thiol-
functional to branched copolymer stabilisers
The functional-emulsions initially envisaged for these studies
require biologically/pharmacologically-relevant oil phases to be
stabilised in water. As such, and building on our recent
24,31
reports,
emulsions were generated using the naturally
occurring polyunsaturated liquid hydrocarbon squalene using
the thiol-functional branched copolymers. This more detailed
study was limited to [SH0.75/Dod0.25]–p(OEGMA50-co-
EGDMA0.80) due to its balance of high thiol content, a predicted
efficient number of Dod chain-ends to maintain emulsication
performance, a good balance of molecular weight, and a weight
fraction of highly branched chains representing a high proba-
bility of thiol content (8 wt% $ 19 chains; 4 wt% $ 28 chains;
and 2 wt% $ 37 conjoined chains) (ESI, Table S6†).
Squalene emulsions were generated under identical condi-
tions to dodecane emulsions described above and, as expected,
Scheme
2
Preparation of thiol functional branched copolymer this [SH0.75/Dod0.25]–p(OEGMA50-co-EGDMA0.80
)
branched
emulsifiers. (a) Incorporation of protected thiols (xanthates) at polymer copolymer acted as a highly efficient emulsier, generating
end-groups via co-initiated ATRP of OEGMA in the presence of
approximately 16 mm oil droplets (D[4,3]) within an aqueous
EGDMA. (b) Thiol deprotection via aminolysis to expose thiol func-
continuous phase (ESI, Table S9†). The potential for disulde
tionalities at pre-determined end-group compositions.
bond formation on storage, leading to possible aggregation or
demulsication, was studied by storing the thiol-functional
emulsion under atmospheric conditions and ambient temper-
ature for extended periods. Laser diffraction measurements
taken each week for 4 weeks showed no meaningful variation or
effect of storage and no visible separation of oil was observed
ꢀ
1
>
(
500 kg mol which equates to >10 conjoined primary chains
ESI, Fig. S22 and Table S6†). This was highly encouraging as the
weight fractions of high molecular weight, highly branched
structures are broadly similar to those shown to act as efficient
emulsiers. Removal of the xanthate protecting group, and
liberation of the thiol-functionality, was conducted for
a selected series of functional copolymers, leading to the
(ESI Fig. S26†). Squalene is a good solvent for a range of mole-
cules including two dye molecules, the diazo dye Oil Red O and
an anthraquinone Oil Blue A, which were studied as models of
future therapeutic agents that may be incorporated into the
emulsions (Fig. 6 & 7). This was achieved via addition of small
amounts (0.1 wt% w.r.t. oil phase) of the respective dye into the
systematically-varying thiol-functional copolymers [SH0.25
Dod0.75]–p(OEGMA -co-EGDMA0.80), [SH0.50/Dod0.50]–
/
50
This journal is © The Royal Society of Chemistry 2020
RSC Adv., 2020, 10, 30463–30475 | 30469

View Article Online
RSC Advances
Paper
Table 2 Triple detection size exclusion chromatography analysis of xanthate-containing (co)polymers
a
ꢀ1
a
ꢀ1
a
Polymer
M
n
(g mol
)
M
w
(g mol
)
Đ
Xan1.00–p(OEGMA50
)
22 800
45 850
38 250
52 000
70 750
37 150
64 800
39 300
1.72
6.48
3.86
7.72
3.98
3.62
5.67
[
[
[
[
[
[
Xan0.08/Dod0.92]–p(OEGMA50-co-EGDMA0.80
)
)
)
)
)
)
297 050
147 500
401 500
281 900
134 500
367 200
Xan0.25/Dod0.75]–p(OEGMA50-co-EGDMA0.80
Xan0.50/Dod0.50]–p(OEGMA50-co-EGDMA0.80
Xan0.75/Dod0.25]–p(OEGMA50-co-EGDMA0.80
Xan0.90/Dod0.10]–p(OEGMA50-co-EGDMA0.80
Xan0.95/Dod0.05]–p(OEGMA50-co-EGDMA0.80
a
ꢁ
TD-SEC using DMF/0.01 M LiBr eluent (60 C); all polymerisations achieved $99% monomer conversion.
emulsication process. Incorporation of the dye molecules the surfactant polysorbate 80 is also present. The biosimilar
resulted in slightly smaller emulsions (D[4,3] approximately 13 mucus was spread evenly across a glass microscope slide to
mm) and samples stabilised with either Dod1.00–p(OEGMA -co- provide
a mucosal surface for investigation by optical
50
EGDMA0.80) or [SH0.75/Dod0.25]–p(OEGMA -co-EGDMA0.80) were
50
generated to investigate the impact of thiol-functionality on the
stable emulsions (ESI, Fig. S27†).
Several biosimilar mucus formulations have been reported;
32
a mimic of natural mucus, outlined by Boegh et al., has been
reported as being rheologically optimised to strongly resemble
natural porcine intestinal mucus. The biosimilar mucus
contains Carbopol 940 (a high molecular weight crosslinked
poly(acrylic acid)), to provide a shear-thinning dominant elastic
behaviour, whilst chemical similarity is obtained by incorpo-
ration of porcine mucin, bovine serum albumin and a lipid
mixture of cholesterol, phosphatidylcholine and linoleic acid;
Fig. 7 Optical microscopy images of emulsions in the presence of
a biosimilar mucosal surface. (A) Emulsion stabilised by [SH0.75
Fig. 6 Optical microscopy images of squalene-in-water emulsions Dod0.25]–p(OEGMA50-co-EGDMA0.80) with Oil Red O (0.1 wt%), (i)
containing Oil Blue (0.1 wt%). (A) [Dod1.00]–p(OEGMA50-co- immediately after application, (ii) after 10 minutes. (B) Emulsions sta-
/
A
EGDMA0.80)- and (B) [SH0.75/Dod0.25]–p(OEGMA50-co-EGDMA0.80)- bilised by [SH0.75/Dod0.25]–p(OEGMA50-co-EGDMA0.80) with Oil Red
stabilised emulsions both (i) before and (ii) 10 minutes after addition to O (top) and Oil Blue A (bottom) (i) immediately after application, (ii)
a biosimilar mucosal surface.
after 10 minutes and (iii) presence of a mixed mobile liquid oil layer.
30470 | RSC Adv., 2020, 10, 30463–30475
This journal is © The Royal Society of Chemistry 2020

View Article Online
Paper
RSC Advances
microscopy; each sample was viewed and monitored closely for
1
0 minutes aer the addition of the concentrated emulsions to
the mucus substrate. Squalene emulsions stabilised with
Dod1.00]–p(OEGMA -co-EGDMA0.80) (Fig. 6A(i)) were unaf-
[
50
fected by the mucus surface, spreading evenly across the
substrate and maintaining their ability to move throughout the
study; ready identication of individual stable droplets was
achieved via optical microscopy, with no observable aggregation
or perturbation of the dispersed oil phase (Fig. 6A(ii)). This
behaviour was in marked contrast to the emulsions stabilised
with [SH0.75/Dod0.25]–p(OEGMA -co-EGDMA0.80) (Fig. 6B);
50
addition of this emulsion to the biosimilar mucus-covered glass
slide led to instantly observable differences in spreading and
aggregation. During the same 10 minute observation period, the
formation of free oil aer droplet rupturing was also seen
(Fig. 6B(ii)).
A separate [SH0.75/Dod0.25]–p(OEGMA50-co-EGDMA0.80)-sta-
bilised squalene-in-water emulsion was prepared, containing
Oil Red O (ESI Fig. S28†). Exchange of Oil Blue A for Oil Red O
had no impact on the mucus-responsive behaviour, with
extensive demulsication occurring within 10 minutes of
addition to a biosimilar mucosal surface (Fig. 7A(i) and (ii)).
The mucus-triggered demulsication was investigated
Fig. 8 Schematic representation of; (A) repulsion between neighbouring
emulsion oil droplets due to the steric hindrance of OEGMA pendant
chains leading to high levels of stability and (B) disulfide bond formation
between the thiol groups present on the surface of the oil droplets and the
further via simultaneous addition of the two dye-loaded, thiol- mucosal surface leading to droplet destabilisation and release of free oil.
functional emulsions to the same mucus substrate. As seen in
previous experiments, the emulsions adhered to the mucus at
the point of addition with a clear boundary between the col-
oured emulsions and limited mixing (Fig. 7B(i)). The resulting
rupturing of the oil phases with each zone of coloured emulsion
rich areas of the porcine mucus are able to bond readily with
thiol-functional emulsion droplets (Fig. 8B).
The demulsication behaviour, and release of free oil,
suggests that the formation of covalent bonds with the mucus
substrate leads to either a removal of branched copolymer sta-
biliser from the oil/water interface, with subsequent destabili-
sation and agglomeration, or the modication of the contact
angle at the mucus–emulsion interface and causing the droplet
to rupture and spread resulting in agglomeration and release of
the emulsied oil.
(
(
Fig. 7B(ii)), led to a mobile liquid oil layer that quickly mixed
Fig. 7B(iii)), generating a purple colour, in the presence of
distinct and static red and blue areas. This suggests opportu-
nities for the isolation of incompatible oil-soluble chemistries,
or combination products, with triggered demuslication and
mixing on target mucosal surfaces.
The limited spreading, and observed lack of motion, of the
[SH0.75/Dod0.25]–p(OEGMA50-co-EGDMA0.80)-stabilised
emul-
sion suggests rapid and strong mucus interactions, presumably
via disulde bond formation between the cysteine-rich regions
within the porcine mucin and the thiol functionality presented
Conclusions
at the oil droplet surface. Chemical bond formation during the Mucoadhesive materials offer a considerable opportunity for
consolidation stage is characteristic of the so-called “second the formulation and delivery of a range of compounds to target
generation” mucoadhesive materials and disulde bond mucosal sites. Mucoadhesive emulsions are of particular
formation is the basis of most thiolated mucoadhesive macro- interest as the ability to dissolve hydrophobic materials within
molecules. This correlates well with the literature “contact/ the stabilised oil-droplets offers the potential to carry active
consolidation” model of mucoadhesion but is surprising given molecules and deliver them to specic locations; antimicro-
the high stability of the thiol-functional droplets in the absence bials, antivirals, hygiene agents and drug compounds are of
of mucus. We hypothesise that the thiols at the branched clear interest but materials able to diagnose, label or indicate
copolymer chain ends are unable to approach each other to local environmental conditions also have applications. Here, we
form bridging disulde bonds upon storage, due to the steric have shown a novel strategy for generating bespoke thiol-
repulsion of the branched p(OEGMA) chains at the oil-droplet/ functional, highly efficient emulsiers and, to the best of our
water interface (Fig. 8A); intra-droplet reaction is also highly knowledge, such materials have not been reported previously.
unlikely given the density of OEGMA side chains along the The branched copolymer synthesis platform, copolymerising
p(OEGMA) backbone. The rapid interaction with the mucus a low concentration of bifunctional monomer within a conven-
substrate appears to suggest that steric repulsion is not a major tional ATRP polymerisation, allows not only the formation of
factor at this interface, and the thiols present in the cysteine- high molecular weight materials, but also the introduction of
amphiphilicity with controlled chain-end functionalisation; the
This journal is © The Royal Society of Chemistry 2020
RSC Adv., 2020, 10, 30463–30475 | 30471

View Article Online
RSC Advances
Paper
ꢁ
demonstration of chain-end redundancy and thiol-functionality aqueous polymer solutions at 20 C. The contact angles of
introduction offers new avenues for functional-emulsion aqueous polymer solution droplets rested on a Teon substrate
research. Mucus-triggered oil release was not an initial target were measured using A Kruss DSA100E drop shape analyser
of this study but the ability to form such stable emulsions with equipped with an advanced digital camera (ꢃ20 magnication).
rapid release is remarkable and requires additional research to
understand both the limitations of the approach and its full
opportunities. For simplicity, one branched copolymer emul-
sier and one ratio of emulsier to oil, at one oil-droplet size, Experimental procedures
were selected for these initial mucus–substrate interaction
Initiator synthesis – 2-dodecyl 2-bromoisobutyrate (Dod-Br); 1
studies, however, there are options to tailor the interactions
1
6
-Dodecanol (9.32 g, 50.0 mmol, 1.00 equiv.) and TEA (6.07 g,
0.0 mmol, 1.20 equiv.) were dissolved in dichloromethane
through concentration of thiol-functional groups and oil-
droplet size; these studies are ongoing.
(
DCM, 70 mL). A solution of a-bromoisobutyl bromide (13.8 g,
0.0 mmol, 1.20 equiv.) in DCM was added dropwise to the
mixture via a pressure equalising dropping funnel and stirred in
an ice bath under a nitrogen (N ) atmosphere. Aer addition,
6
Materials and methods
Materials
2
the reaction vessel was le to warm to ambient temperature and
was le stirring for 24 hours. The solution was washed with
Calcium chloride (CaCl ), magnesium sulfate (MgSO ), sodium
2
4
hydrogen carbonate (NaHCO ), sodium chloride (NaCl), trie-
3
NaHCO
3
(1 M, 1 ꢃ 50 mL) and deionised water (4 ꢃ 50 mL). The
thylamine (TEA) and hydrochloric acid (HCl, 1 M) were
purchased from Fisher Scientic and used as received. Ethylene
glycol (99.8%), anhydrous tetrahydrofuran (THF, 99%), 1-
dodecanol (99%), a-bromoisobutyl bromide (98%), anisole
organic layer was then dried over anhydrous MgSO , ltered,
4
and concentrated in vacuo. The product was then passed
through a basic alumina column. The product, 2-dodecyl 2-
bromoisobutyrate (Dod-Br); 1, was isolated as a clear oil in 53%
(
99%), oligo(ethylene glycol methacrylate) (OEGMA, M
n
¼ 300 g
1
yield (8.91 g) and the correct structure was conrmed using H
ꢀ
1
0
mol , 98%), ethylene glycol dimethacylate (EGDMA), 2,2 -
bipyridyl (bpy), copper(I) chloride (CuCl, 97%), activated neutral
alumina, basic alumina, porcine mucin, bovine serum albumin
1
3
1
and C NMR spectroscopy (ESI Fig. S1†). H NMR (400 MHz,
CDCl
) d (ppm) ¼ 0.81 (t, J ¼ 6.6 Hz, 3H), 1.19 (m, 18H), 1.61 (m,
H), 1.86 (s, 6H, CH
3
1
3
2
3
), 4.08 (t, J ¼ 6.6 Hz, 2H). C NMR (100
(BSA, >98%), cholesterol, linoleic acid, phosphatidylcholine and
MHz, CDCl ) d (ppm) ¼ 14.1, 22.7, 25.8, 28.4, 29–30, 32.0, 56.0,
3
polysorbate 80 were purchased from Sigma Aldrich and used as
received. Potassium ethyl xanthogenate (96%), 2-bromoacetic
+
6
3
6.2, 171.8. Mass spectrometry: [MH] (C H BrO ) m/z ¼
1
6
32
2
+
35.0. Experimentally: ES MS [MH] m/z ¼ 335.2. Elemental
Br) ¼ C, 57.31; H, 9.32.
Experimental (%) ¼ C, 57.33; H, 9.20.
0
acid, 4-(dimethylamino) pyridine (DMAP, >99%), N,N -dicyclo-
31 2
analysis: calculated (%): (C16H O
hexylcarbodiimide (DCC, >99%) and 2-bromoacetic acid were
all purchased from Alfa Aesar and used as received. 4-
(
Dimethylamino)pyridinium-4-toluene sulfonate (DPTS) was
25
synthesised as previously reported. Polyacrylic acid (Carbopol
40) was purchased from Lubrizol. All solvents, unless stated
9
Initiator synthesis – 2-poly(ethylene glycol)17 2-
otherwise, were reagent grade, purchased from Fisher Chem-
icals and used as received.
bromoisobutyrate (PEG -Br); 2
1
7
ꢀ
1
Monomethoxy poly(ethylene glycol) (M ¼ 750 g mol , 23.0 g,
n
ꢁ
30.7 mmol, 1.00 equiv.) was dissolved in anhydrous THF (40 C)
Characterisation
2
under a dry N atmosphere. DMAP (0.038 g, 0.30 mmol, 0.01
Nuclear magnetic resonance (NMR) spectroscopy was recorded equiv.) and TEA (5.60 g, 53.7 mmol, 1.75 equiv.) were added and
using a Bruker DPX-400 spectrometer operating at 400 MHz for the reaction mixture stirred in an ice bath. a-Bromoisobutyrl
1
13
H and 100 MHz C nuclei respectively. Molecular weight data bromide (10.60 g, 46.0 mmol, 1.51 equiv.) was added dropwise
was obtained by triple detection size exclusion chromatography using a dropping funnel over 30 minutes and a white precipitate
+
ꢀ
(TD-SEC) using a Malvern Viscotek SECMax. The instrument was (Et NH Br salt) was immediately formed. The reaction was le
3
equipped with a GPCmax VE2001 auto sampler, two Viscotek for 24 hours, the precipitate removed by ltration and the
D6000 columns, a guard column and a triple detector array solvent removed in vacuo; the resulting crude product was
TDA305 (refractive index, light scattering and viscometer). TD- precipitated twice from acetone into petroleum ether (30–40
ꢁ
ꢁ
SEC was conducted at 60 C using a mobile phase of DMF
containing 0.01 M lithium bromide (LiBr) at a ow rate of 1 (PEG17-Br); 2, was isolated as a white wax in 65% yield (15.0 g)
C). The product, 2-(polyethylene glycol)17 2-bromoisobutyrate
ꢀ1
1
13
mL min . Emulsications were conducted using an IKA T25 and the correct structure was conrmed using H and C NMR
ULTRA-TURRAX high-shear homogeniser. Oil-in-water (O/W) spectroscopy (ESI Fig. S2†). H NMR (400 MHz, D
emulsions were characterised by laser diffraction using a Mal- 1.86 (s, 6H), 3.30 (S, 3H), 3.64 (m, 60H), 3.77 (m, 2H), 4.32 (m,
1
2
O) d ppm ¼
1
3
vern Mastersizer 2000A equipped with a Hydro 2000 SM 2H). C NMR (100 MHz, D O) d ppm ¼ 29.8, 56.8, 65.4, 38.3,
2
dispersion unit. Microscope images were obtained using a Leica 69.5, 69.5, 69.7, 70.9, 173.7. Elemental analysis: calculated (%):
DM4 B microscope tted with a CMOS camera. A Kibron Delta-8 (C37
surface tensiometer was used to measure the surface tension of H, 8.31.
H
73
O
18Br) ¼ C, 50.16; H, 8.24. Experimental (%) ¼ C, 49.25;
30472 | RSC Adv., 2020, 10, 30463–30475
This journal is © The Royal Society of Chemistry 2020

View Article Online
Paper
RSC Advances
Initiator synthesis – 2-(2-((ethoxycarbonothioyl)thio)acetoxy)
ethyl 2-bromoisobutyrate (Xan-Br), 3
was ltered, diluted in dichloromethane (100 mL) and washed
with deionised water (2 ꢃ 100 mL) and brine (1 ꢃ 100 mL). The
organic layer was dried over MgSO . Aer the removal of DCM in
4
The synthesis of 2-(2-((ethoxycarbonothioyl)thio)acetoxy)ethyl 2-
bromoisobutyrate (Xan-Br); 3, required the preparation of two
precursor molecules; 2-((ethoxycarbanothioyl)thio) acetic acid
and 2-hydroxyethyl 2-bromoisobutyrate. For the synthesis of 2-
vacuo, further purication was performed by automated liquid
chromatography (silica gel, eluting a gradient mobile phase
increasing in polarity from hexane to hexane : ethyl acetate
(70 : 30)). The product, 2-(2-((ethoxycarbonothioyl)thio)acetoxy)
((ethoxycarbanothioyl)thio) acetic acid, potassium ethyl xan-
ethyl 2-bromoisobutyrate, Xan-Br; 3, was isolated as a yellow oil
in 70% yield (5.08 g) and the correct structure was conrmed
thogenate (53.1 g, 311.0 mmol, 1.0 equiv.) was added to a two-
neck round bottom ask and stirred in acetone (400 mL). A
solution of 2-bromoacetic acid (38.4 g, 276.0 mmol, 0.08 equiv.)
in acetone (100 mL) was added dropwise to the reaction vessel
over 20 minutes using a dropping funnel and the reaction was
le to stir at ambient temperature for 16 hours. The crude
mixture was ltered under vacuum, the residue washed with
acetone and the solvent removed in vacuo. The residual oil was
solubilised in a minimum amount of dichloromethane and
washed with brine (150 mL). The organic layer was dried over
1
13
1
using H and C NMR spectroscopy (ESI Fig. S5†). H NMR (400
MHz, CDCl ) d ppm ¼ 1.43 (t, J ¼ 7.1 Hz, 3H), 1.94 (s, 6H), 3.96
3
1
3
(
s, 4H), 4.41 (s, 4H), 4.66 (q, J ¼ 7.1 Hz, 2H). C NMR (100 MHz,
CDCl
2
3
) d ppm ¼ 13.70, 30.7, 37.7, 63.0, 63.3, 70.8, 167.8, 171.5,
+
12.5. Mass spectrometry calculated: [MNa] (C11
H
17BrO
m/z ¼ 395.28. Experimental: ES MS [MNa] m/z ¼ 395. Elemental
analysis: calculated (%): (C11 17BrO
) ¼ C, 35.39; H, 4.59; S,
7.18. Experimental (%) ¼ C, 36.29; H, 4.79; S, 17.06.
5 2
S Na)
+
H
5 2
S
1
MgSO and the solvent was removed in vacuo to give a white
solid. The product, 2-((ethoxycarbanothioyl)thio) acetic acid,
4
Polymer synthesis – linear polymerisations
was isolated in 33% yield (16.28 g) and the correct structure was For the synthesis of linear p(OEGMA) samples, targeting DP ¼
n
1
13
conrmed using H and C NMR spectroscopy (ESI Fig. S3†). 50 monomer units, Dod-Br (0.027 g, 0.08 mmol, 1.00 equiv.),
1
H NMR (400 MHz, CDCl
3
) d ppm ¼ 1.43 (t, J ¼ 7.1 Hz, 3H), 3.98 PEG -Br (0.25 g, 0.34 mmol, 1.00 equiv.) or Xan-Br, (0.13 g,
1
7
13
0
(
s, 2H), 4.65 (q, J ¼ 7.1 Hz, 2H), 9.3 (s, br, –OH). C NMR (100 0.30 mmol, 1.00 equiv.) along with 2,2 -bipyridyl (0.10 g,
MHz, CDCl ) d ppm ¼ 13.70, 37.63, 70.93, 173.86, 212.07. Mass 0.64 mmol, 2.00 equiv.) and OEGMA (5.00 g, 16.0 mmol, 50.0
3
+
spectrometry: calculated: [MH] (C H BrO ) m/z ¼ 211.0. equiv.) were added to a 25 mL round bottom ask, equipped
6
12
3
+
Experimental: CI MS [MNa] m/z ¼ 211.0. Elemental analysis: with a magnetic stirrer bar. The mixture was deoxygenated via
calculated (%): (C
5
H
8
O
3
S
2
) ¼ C, 33.32; H, 4.47; S, 35.58. Exper- N2 bubbling for 30 minutes. A solvent system of IPA/H O
2
imental (%) ¼ C, 32.80; H, 4.40; S, 34.59.
(92.5 : 7.5 v/v%, 4.39 : 0.36 mL, 55 wt% w.r.t to monomer,
For the synthesis of 2-hydroxyethyl 2-bromoisobutyrate, deoxygenated separately via N purge) was added to the reaction
2
ethylene glycol (301 g, 4.86 mol, 50.0 equiv.) and TEA (20.3 g, vessel and the mixture was deoxygenated by N bubbling for
2
0.20 mol, 2.00 equiv.) were dissolved in anhydrous tetrahydro- a further 5 minutes. Anisole (ca. 100 mL) was added to the
furan (100 mL) and the reaction was stirred in an ice bath. a- reaction ask for use as an internal standard for determination
1
Bromoisobutyl bromide (22.32 g, 97.1 mmol, 1.00 equiv.) was of monomer conversion by H NMR. This was calculated from
added dropwise over 30 minutes and the reaction was le stir- the change in the intensity of OEGMA vinyl peaks during poly-
1
ring under nitrogen atmosphere at ambient temperature for 16 merisation, relative to those of the anisole aromatic H reso-
hours. The reaction mixture was poured into deionised water nances (ESI, Fig. S6†). CuCl (0.032 g, 0.32 mmol, 1.00 equiv.)
(
800 mL) and extracted with dichloromethane (6 ꢃ 100 mL), the was added and the reaction vessel was then sealed. The ask
ꢁ
retrieved layers were washed with 1 M HCl (2 ꢃ 300 mL), dried was placed into a pre-heated oil bath (40 C) and le for 24 h
over MgSO
2
4
and the solvent was removed in vacuo. The product, aer which the polymerisation was stopped by cooling the ask
-hydroxyethyl 2-bromoisobutyrate, was isolated as a clear oil in to ambient temperature, exposure to oxygen and dilution with
1% yield (18.6 g) and the correct structure was conrmed using THF. A neutral alumina column was used to remove the copper
9
1
13
1
H and C NMR spectroscopy (ESI Fig. S4†). H NMR (400 MHz, catalyst, and the solvent was removed in vacuo. The crude
CDCl
3
) d ppm ¼ 1.96 (s, 6H), 3.87 (t, J ¼ 4.7 Hz, 2H), 4.31 (t, J ¼ polymer was puried by precipitation (twice) from acetone into
1
3
ꢁ
4
6
.7 Hz, 2H). C NMR (100 MHz, CDCl
3
) d ppm ¼ 30.7, 55.8, cold petroleum ether (30–40 C). Residual solvent was removed
ꢁ
0.7, 63.3, 67.4, 171.8. Elemental analysis: calculated (%) ¼ in a vacuum oven at 40 C overnight. The puried polymers
1
(C
6
H
11BrO
3
) ¼ C, 34.14; H, 5.25. Experimental (%) ¼ C, 34.09; H, were characterised by H NMR in CDCl3 and TD-SEC using
5.24.
a DMF/LiBr (0.01 M) eluent.
For the synthesis of 3, 2-((ethoxycarbanothioyl)thio) acetic
acid (3.85 g, 21.2 mmol, 1.00 equiv.), 2-hydroxyethyl 2-bromoi-
sobutyrate (4.50 g, 21.2 mmol, 1.00 equiv.) and DPTS (6.86 g,
Polymer synthesis – branching polymerisations
All branching copolymerisations were conducted using
a constant molar ratio of branching co-monomer to initiator of
2
3.3 mmol, 1.10 equiv.) were dissolved in anhydrous dichloro-
methane (40 mL) under a N atmosphere. DCC (4.81 g,
3.3 mmol, 1.10 equiv.) was dissolved in anhydrous dichloro-
methane (10 mL) and transferred, under a N ow, to the main
2
0
.80 to 1.00 ([EGDMA]
0
/[I]
0
¼ 0.80) where I ¼ Dod-Br or PEG17-Br
2
or Xan-Br. For co-initiated copolymerisations – for example in
the preparation of [Dod /PEG( ) ]–p(OEGMA -co-EGDMA0.80)
2
x
17 y
50
reaction vessel using a syringe and the reaction was le to stir at
ambient temperature for 16 hours. The resulting crude mixture
P
or [Dod
x
/Xan
y
]–p(OEGMA50-co-EGDMA0.80), where [I]
0
¼ x + y
¼
1.00 equivalents – initial molar initiator ratios of [Dod-
This journal is © The Royal Society of Chemistry 2020
RSC Adv., 2020, 10, 30463–30475 | 30473

View Article Online
RSC Advances
Br] : [PEG17-Br]
Paper
0
0
or [Dod-Br]
0
: [Xan-Br]
0
were systematically was prepared in a round bottom ask (10 mL) equipped with
varied to give branched polymers containing controlled chain- a magnetic stirrer bar. The solution was studied through three
end compositions of 1.00 : 0.00, 0.90 : 0.10, 0.75 : 0.25, heating/cooling cycles in an oil bath at a heating/cooling rate of
ꢁ
ꢀ1
0.50 : 0.50, 0.25 : 0.75, 0.10 : 0.90 and 0.00 : 1.00.
approximately 1 C min . The temperatures of copolymer
In a typical mixed initiated synthesis of [Dod
x
/PEG(17)y]– solutions were monitored until the cloud point was observed,
p(OEGMA50-co-EGDMA0.80), targeting DP
n
¼ 50 monomer units, i.e. when the transparent solution turned opaque, indicating
P
where [I]
0
¼ x + y ¼ 0.25 + 0.75 ¼ 1.00 equiv., OEGMA (5.00 g, precipitation of the dissolved macromolecules. LCST values
16.0 mmol, 50.0 equiv.), EGDMA (0.051 g, 0.32 mmol, 0.80 were quoted as the mean value (n ¼ 3 ꢂ standard deviations).
0
equiv.), 2,2 -bipyridyl (0.100 g, 0.64 mmol, 2.00 equiv.), Dod-Br
(
0.027 g, 0.08 mmol, 0.25 equiv.), PEG -Br (0.19 g,
17
Characterisation – surface tensiometry measurements
0
.25 mmol, 0.75 equiv.) and a solvent system of IPA/H O
2
Aqueous copolymer solutions were prepared at a concentration
(
92.5 : 7.5 v/v, 4.39 : 0.36 mL, 55 wt% w.r.t to monomer, deox-
ygenated separately via a N purge) were added to a RBF
equipped with a magnetic stirrer bar and all components were
deoxygenated via N bubbling for 30 minutes. Anisole (ca. 100
of 30 wt% in deionised (DI) H
2
O. Serial dilutions of such
2
copolymer solutions in DI H O were used to yield 30 different
2
ꢀ
9
copolymer concentrations ranging from 6.9 ꢃ 10 to 30 wt%.
The use of a 96 well-plate allowed for high throughput analysis
of samples; 50 mL of each polymeric surfactant (n ¼ 8) was used
per well and measured against pure water as the control.
Surface tension data was plotted against a log concentration of
the surfactant. Critical micelle concentrations (CMC) were
calculated by determining the intercept between the lines of
best t obtained from the linear decreases in surface tension
2
mL) was added to the reaction mixture for use as an internal
standard for calculation of monomer conversions by H NMR.
CuCl (0.032 g, 0.32 mmol, 1.00 equiv.) was then added, under
a positive pressure of N , and the reaction vessel was sealed.
Polymerisation and purication procedures were conducted in
accordance with the method used for linear polymerisations,
described above.
1
2
(slope) and the lower plateau area.
Polymer synthesis – preparation of thiolated branched
copolymers via co-initiated ATRP
Characterisation – contact angle measurements
Aqueous copolymer solutions (5 wt%) were prepared and
applied to a glass microscope slide covered with a polytetra-
In
a
typical synthesis of [Xan
x
/Dod
y
]–p(OEGMA50-co-
P
EGDMA0.80), targeting DP
n
¼ 50 monomer units, where [I]
0
¼

uoroethylene (PTFE) substrate, to create a hydrophobic
x + y ¼ 0.75 + 0.25 ¼ 1.00 equiv., OEGMA (5.00 g, 16.0 mmol,
surface. Aqueous copolymer solutions (5 mL) were applied to the
slide and contact angles measured using the static sessile drop
method (n ¼ 10 ꢂ standard deviations).
0
50.0 equiv.), EGDMA (0.051 g, 0.32 mmol, 0.80 equiv.), 2,2 -
bipyridyl (0.100 g, 0.64 mmol, 2.00 equiv.), Dod-Br (0.027 g,
.08 mmol, 0.25 equiv.), Xan-Br (0.089 g, 0.24 mmol, 0.75 equiv.)
0
were added to a RBF equipped with magnetic stirrer and solvent
Characterisation – preparation of biosimilar mucus
system IPA/H O (92.5 : 7.5 v/v, 4.39 : 0.36 mL, 55 wt% wrt to
2
monomer, deoxygenated separately via N
Anisole (ca. 100 mL) was added to the reaction for use as an buffer solution (9.00 mL, 1.30 mM CaCl
2
purge) was added. Carbopol® 940 (0.90 g, 0.90 w/v%) was dissolved in HEPES
, 1.00 mM MgSO and
2
4
1
internal standard for calculation of monomer conversion by H 137 mM NaCl, pH 7.4) using magnetic stirring. A lipid mixture
NMR. CuCl (0.032 g, 0.32 mmol, 1.00 equiv.) was added and the of phosphatidylcholine (0.018 g, 0.18% weight/volume (w/v)%),
reaction vessel was sealed. Polymerisation and purication cholesterol (0.0036 g, 0.36 w/v%), polysorbate 80 (0.033 g, 4 : 1
processes were conducted in accordance with the method used ratio) and HEPES buffer solution (1 mL) was prepared and
for linear polymerisations, described above. In a typical added to the Carbopol solution when approximately 90% of the
deprotection procedure to yield [SH0.75/Dod0.25]–p(OEGMA -co- Carbopol had dissolved. Mucin from porcine stomach (0.50 g,
50
EGDMA0.80), the puried copolymer, [Xan0.75/Dod0.25]– 5.00 w/v%) was added to the solution and the pH altered to 7.4
p(OEGMA50-co-EGDMA0.80) (0.60 g, 1.66 mmol, 1.00 equiv.) and with the addition of NaOH (1 M), which was monitored using
anhydrous THF (10 mL) were added to a 25 mL round bottom a pH probe. BSA (0.31 g, 3.10 w/v%) was added and the pH was

ask equipped with a magnetic stirrer bar. The ask was sealed once again adjusted to 7.4. Biosimilar mucus was stored over-
ꢁ
and deoxygenated via a N
2
purge for 10 minutes. n-Butylamine night at 2–4 C before use and discarded if not used within
(0.38 mL, 4.15 mmol, 2.50 equiv.) was then added and the a maximum of four days from the day of preparation.
reaction was le to stir at ambient temperature. Aer 1.5 hours,
the solvent was removed in vacuo and the crude polymer
precipitated from acetone into hexane (twice). The pure copol-
Preparation and characterisation of O/W emulsions
O/W emulsions were prepared using a 1 : 1 v/v ratio of oil-
ꢁ
ymer was then dried in a vacuum oven at 40 C for 48 hours.
:
water, using either n-dodecane or squalene as the oil phase.
ꢀ
1
Aqueous copolymer solutions (3 mL, 5 mg mL ) and n-
dodecane or squalene (3 mL) were added to a glass vial (14 mL);
the mixture was then homogenised using an IKA T 25 ULTRA-
Characterisation – lower critical solution temperature
measurements
For a typical lower critical solution temperature (LCST) TURRAX over-head high-shear homogeniser at 24 000 rpm for
measurement, an aqueous copolymer solution (5 mL, 1 wt%) 2 minutes. Emulsions were stored overnight in a sealed glass
30474 | RSC Adv., 2020, 10, 30463–30475
This journal is © The Royal Society of Chemistry 2020

View Article Online
Paper
RSC Advances
vial at ambient temperature before characterisation via laser
diffraction. O/W emulsions were added dropwise to the
dispersion unit containing approximately 100 mL deionised
8 J. D. Smart, Adv. Drug Delivery Rev., 2005, 57, 1556–1568.
9 S. Mansuri, P. Kesharwani, K. Jain, R. K. Tekade and
N. K. Jain, React. Funct. Polym., 2016, 100, 151–172.
water (DI H O) at a stirring rate of 1000 rpm at ambient 10 A. Bernkop-Schn u¨ rch and A. Greimel, Am. J. Drug Delivery,
2
temperature. The volume-average droplet diameters (D[4,3]) are
2005, 3, 141–154.
quoted as an average of $20 measurements, where D[4,3]
¼
11 R. Kumar and V. R. Sinha, React. Funct. Polym., 2013, 73,
1156–1166.
P
P
4
3
D
i
N
i
/
D
i
N
i
. For further stability studies, emulsions were
stored in sealed glass vials at ambient temperature and were re- 12 H. T. Lam, G. Leonaviciute, O. Zupan ˇc i ˇc and A. Bernkop-
assessed by laser diffraction and optical microscopy at the time
intervals stated.
Schn u¨ rch, Eur. J. Pharm. Sci., 2017, 106, 41–48.
13 B. Chatterjee, N. Amalina, P. Sengupta and U. K. Mandal, J.
Appl. Pharm. Sci., 2017, 7, 195–203.
Mucosal trigged emulsion release studies
14 V. M. Leitner, G. F. Walker and A. Bernkop-Schn u¨ rch, Eur. J.
Pharm. Biopharm., 2003, 56, 207–214.
For mucosal triggered release studies emulsions were prepared
as above with the inclusion of either Oil Red O or Oil Blue A
´
´
´
´
1
5 B. A. Szilagyi, B. Gyarmati, G. Horvat, A. Laki,
M. Budai-Sz u} cs, E. Cs ´a nyi, G. Sandri, M. C. Bonferoni and
A. Szil ´a gyi, Polym. Int., 2017, 66, 1538–1545.
(0.1 wt% with respect to the oil phase) as a hydrophobic drug
mimic. In a typical mucosal triggered release study, biosimilar
mucus was spread onto a glass microscope slide. Polymeric
emulsier-stabilised O/W emulsions (100 mL) were applied to
the centre of the glass slide. The slides were then imaged using
optical microscopy both immediately aer application of the O/
W emulsion and again 10 minutes aer the addition.
1
1
1
1
2
6 G. Shahnaz, G. Perera, D. Sakloetsakun, D. Rahmat and
A. Bernkop-Schn u¨ rch, J. Controlled Release, 2010, 144, 32–38.
7 M. Davidovich-Pinhas, O. Harari and H. Bianco-Peled, J.
Controlled Release, 2009, 136, 38–44.
8 M. H. Dufresne, M. A. Gauthier and J. C. Leroux, Bioconjugate
Chem., 2005, 16, 1027–1033.
9 Y. Yu, M. Huo, Y. Fu, W. Xu, H. Cai, L. Yao, Q. Chen, Y. Mu,
J. Zhou and T. Yin, Mol. Pharm., 2017, 14, 4539–4550.
0 H.-B. Lee, S.-Y. Yoon, B. Singh, S.-H. Oh, L. Cui, C. Yan,
S.-K. Kang, Y.-J. Choi and C.-S. Cho, Tissue Eng. Regener.
Med., 2018, 15, 1–11.
Conflicts of interest
SEE and SPR are co-inventors of a recently led patent appli-
cation that describes the work reported here.
2
1 S. Bonengel, S. Hauptstein, G. Leonaviciute, J. Griessinger
and A. Bernkop-Schn u¨ rch, Eur. J. Pharm. Sci., 2015, 75,
Acknowledgements
1
23–130.
The authors are grateful for funding provided by the University
of Liverpool and the Engineering and Physical Sciences
Research Council (EP/R024804/1 and EP/L02635X/1) that sup-
ported part of this research and for PhD studentships (SEE and
SF). SF would also like to thank the University of Liverpool John
Lennon memorial scholarship for nancial support during his
PhD studentship. We also acknowledge the Centre for Materials
Discovery and the Materials Innovation Factory at the University
of Liverpool for access to essential equipment.
2
2
2 J. V. M. Weaver, S. P. Rannard and A. I. Cooper, Angew.
Chem., Int. Ed., 2009, 121, 2165–2168.
3 R. T. Woodward, R. A. Slater, S. Higgins, S. P. Rannard,
A. I. Cooper, B. J. L. Royles, P. H. Findlay and
J. V. M. Weaver, Chem. Commun., 2009, 3554–3556.
4 J. J. Hobson, S. Edwards, R. A. Slater, P. Martin, A. Owen and
S. P. Rannard, RSC Adv., 2018, 8, 12984–12991.
5 J. S. Moore and S. I. Stupp, Room temperature
polyesterication, Macromolecules, 1990, 23, 65–70.
6 S. Flynn, A. B. Dwyer, P. Chambon and S. Rannard, Polym.
Chem., 2019, 10, 5103–5115.
2
2
2
Notes and references
1
2
3
4
C. Atuma, V. Strugala, A. Allen and L. Holm, Am. J. Physiol.: 27 H. Cauldbeck, M. Le Hellaye, M. Long, S. M. Kennedy,
Gastrointest. Liver Physiol., 2001, 280, G922–G929.
M. E. V. Johansson, H. Sj ¨o vall and G. C. Hansson, Nat. Rev.
Gastroenterol. Hepatol., 2013, 10, 352–361.
R. L. Williams, V. R. Kearns and S. P. Rannard, J.
Controlled Release, 2016, 244, 41–51.
28 S. Graham, S. P. Rannard, P. A. G. Cormack and
D. C. Sherrington, J. Mater. Chem., 2007, 17, 545–552.
29 F. L. Hatton, P. Chambon, T. O. McDonald, A. Owen and
S. P. Rannard, Chem. Sci., 2014, 5, 1844.
N. Sharma, S. Mishra, S. Sharma, R. D. Deshpande and
R. Kumar Sharma, Int. J. Drug Dev. Res., 2013, 5, 37–48.
Y. Singh, J. G. Meher, K. Raval, F. A. Khan, M. Chaurasia,
N. K. Jain and M. K. Chourasia, J. Controlled Release, 2017, 30 F. Y. Hern, A. Hill, A. Owen and S. P. Rannard, Polym. Chem.,
52, 28–49.
2018, 9, 1767–1771.
D. J. McClements and S. M. Jafari, Adv. Colloid Interface Sci., 31 F. Y. Hern, S. E. R. Auty, O. C. J. Andr ´e n, M. Malkoch and
017, 251, 55–79.
S. P. Rannard, Polym. Chem., 2017, 8, 1644–1653.
B. Demouveaux, V. Gouyer, F. Gottrand, T. Narita and 32 M. Boegh, S. G. Baldursdottir, A. Mullertz and H. M. Nielsen,
2
5
6
7
2
J.-L. Desseyn, Adv. Colloid Interface Sci., 2018, 252, 69–82.
S. Oh and S. Borr ´o s, Colloids Surf., B, 2016, 147, 434–441.
Eur. J. Pharm. Biopharm., 2014, 87, 227–235.
This journal is © The Royal Society of Chemistry 2020
RSC Adv., 2020, 10, 30463–30475 | 30475