Copolymerization of an indazole ligand into the self-polymerization of dopamine for enhanced binding with metal ions

Article abstract of DOI:10.1039/c5tb01150g

5,6-Dihydroxy-1H-indazole (DHI) is able to self-polymerize through the same mussel-inspired chemistry responsible for generating poly(dopamine) (PDA), demonstrating the potential to expand this class of catecholamine-exclusive chemistry onto heterocyclic catechol derivatives for the preparation of functional materials. Although DHI exhibits slower polymerization kinetics compared to dopamine, the two chemical species are compatibly polymerizable under the same reaction conditions and allow the preparation of copolymer coatings in different molar ratios. Of these copolymers, the 1:3-copolymer (DHI-to-dopamine ratio) has demonstrated adequate structural stability as a polymer coating. While PDA performs as an intact framework, the incorporated DHI enhances the colloidal stability and provides additional coordinating functionality through the pyrazole moieties. The 1:3-copolymer was fabricated into polymer capsules which exhibit negligible cytotoxicity towards murine dermal fibroblasts (L929) and enhanced binding behaviour towards copper(ii). This represents a new channel for fabricating cargo carriers for biomedical applications that involve the use of transition metal-based species.

Full text of DOI:10.1039/c5tb01150g

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Materials Chemistry B
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Copolymerization of an indazole ligand into the
self-polymerization of dopamine for enhanced
binding with metal ions†
Cite this:DOI: 10.1039/c5tb01150g
a
b
b
c
Ka Wai Fan, Justine J. Roberts, Penny J. Martens, Martina H. Stenzel and
a
Anthony M. Granville*
5
,6-Dihydroxy-1H-indazole (DHI) is able to self-polymerize through the same mussel-inspired chemistry
responsible for generating poly(dopamine) (PDA), demonstrating the potential to expand this class of
catecholamine-exclusive chemistry onto heterocyclic catechol derivatives for the preparation of
functional materials. Although DHI exhibits slower polymerization kinetics compared to dopamine, the
two chemical species are compatibly polymerizable under the same reaction conditions and allow the
preparation of copolymer coatings in different molar ratios. Of these copolymers, the 1 : 3-copolymer
(DHI-to-dopamine ratio) has demonstrated adequate structural stability as a polymer coating. While PDA
Received 15th June 2015,
Accepted 25th August 2015
performs as an intact framework, the incorporated DHI enhances the colloidal stability and provides
additional coordinating functionality through the pyrazole moieties. The 1 : 3-copolymer was fabricated
into polymer capsules which exhibit negligible cytotoxicity towards murine dermal fibroblasts (L929) and
enhanced binding behaviour towards copper(II). This represents a new channel for fabricating cargo
carriers for biomedical applications that involve the use of transition metal-based species.
DOI: 10.1039/c5tb01150g
www.rsc.org/MaterialsB
particles, renders facile preparation of PDA-based surface-functional
polymeric capsules.
Through the aqueous-based redox-facilitated self-polymerization
Introduction
2
1–24
Poly(dopamine) (PDA) has stimulated a vast amount of research
based on the mussel-inspired chemistry since it was first reported technique, novel materials have been prepared from a range of
1
25,26
by Lee et al. PDA is highly expressed in catechol and amino monomers, such as norepinephrine,
L-3,4-dihydroxy-phenyl-
2
7,28
29–34
functionalities that mimic the structure of the adhesive protein of alanine (L-DOPA),
mussels and allow the polymer to coat on virtually any material as well as pyrogallol and natural polyphenol compounds,
other synthetic dopamine derivatives,
3
5
with different shapes and geometries. The coated surface is which demonstrated additional functionalities over PDA. These
chemically versatile and capable of secondary modifications, monomers share the same catecholamine structure with the
which makes PDA an ideal surface-functionalizing agent for a ability to form a quinone intermediate and the subsequent cyclized
3
6–38
range of applications in membrane operation/pollution treat- dopamine – 5,6-dihydroxy-indole (DHIn) – when oxidized.
2
–5
6
7–9
ment, microfluidics, micropatterning, and the preparation Further oxidation activates the C-4, C-7 and, to a lesser extent, C-2
1
0–12
of polymer brushes.
platforms for substrate encapsulation.
have actively been researched of late for biomedical applications due
PDA coatings also serve as versatile positions to form the mildly cross-linked, base structure of PDA
13–15
39
PDA-coated substrates (Scheme 1a).
Inspired by the structure of DHIn, we recognized an opportunity
to diverge the development of mussel-inspired self-polymerization
16–20
to a host of desirable properties of this biomimetic polymer,
including negligible cytotoxicity. Removal of the substrates, e.g. silica by substituting dopamine/DHIn with other heterocyclic catechol
derivatives which are also capable to form an activated quinone
structure via the mussel-inspired chemistry. The catecholic
a
indazole – 5,6-dihydroxy-1H-indazole (DHI) – is of substantial
interest because of its pyrazole fused ring, which provides a
Centre for Advanced Macromolecular Design, School of Chemical Engineering,
UNSW, Australia. E-mail: a.granville@unsw.edu.au
b
c
Graduate School of Biomedical Engineering, UNSW, Australia
Centre for Advanced Macromolecular Design, School of Chemistry, UNSW,
Australia
permanent coordination site, i.e. the tertiary amine, in contrast
40–42
to the pyrrole fused ring of DHIn.
Whereas no indazole-
based homopolymer has been prepared previously by this self-
polymerization process, our group has recently demonstrated a
proof-of-concept by integrating a monohydroxy-substituted
†
Electronic supplementary information (ESI) available: UV-Vis, ATR-FTIR, and
NMR spectra, TGA thermograms and experimental procedures. See DOI: 10.1039/
c5tb01150g
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silica particles were purchased from Evonik. Sodium hydrogen
carbonate, magnesium sulfate (dried), copper(II) sulfate penta-
hydrate, and ethyl acetate were purchased from Ajax Finechem
Pty Ltd. Ethanol (95%) was purchased from VWR International.
Dichloromethane (stabilized with amylene) was purchased
from Chem-Supply Pty Ltd. Deionized water was obtained from
a Milli-Q water deionizing unit. All chemicals and solvents were
used as received unless specified otherwise.
5
,6-Dihydroxy-1H-indazole (DHI) monomer synthesis
The DHI monomer was synthesized by demethylating 5,6-dimethoxy-
H-indazole according to the procedure adapted from Schumann
1
60
et al. in consideration of the reagent ratio suggested by Vickery
61
et al. Detailed procedures for the syntheses of precursor com-
pounds are referenced in the ESI.† A solution of 5,6-dimethoxy-1H-
indazole (1 g, 5.61 mmol) in dichloromethane (17.2 mL) was chilled
over an ice-water bath with a nitrogen blanket applied on top.
A solution of boron tribromide (1.0 M in dichloromethane,
Scheme 1 Polymerization of dopamine (a) and DHI (b), and preparation
of hollow capsules from the copolymer (c).
4
3
indazole into the structure of PDA as a chain terminator. DHI 18.5 mL, 18.5 mmol) was added dropwise to the reaction
is an even better prospective candidate for self-polymerization, mixture. The vessel was then capped and the reaction mixture
because of the integrated catechol functionality making it was stirred at room temperature for 4 h. Upon completion, the
capable of behaving as dopamine/DHIn (Scheme 1b).
vessel was submerged in an ice bath and deionized water was
Herein, we report the self-polymerization of DHI based on carefully added to the reaction mixture until the bubbling
the mussel-inspired chemistry. In addition to the homopolymer ceased. The mixture was then neutralized by the slow addition
formation, copolymerization has been proven achievable with of saturated sodium hydrogen carbonate solution. The resulting
mixtures of DHI and dopamine in various molar ratios. The mixture was extracted with dichloromethane, and the aqueous
copolymers represent the opportunity to incorporate specific phase was collected. The product was extracted from the collected
coordination sites, i.e. the pyrazole ring, into the structure of aqueous phase with ethyl acetate. The organic phase was combined
PDA and extend the availability of ligands beyond the catechol and dried over anhydrous magnesium sulfate. Solvent was removed
4
4–46
group.
Moreover, the incorporation of the pyrazole moieties under reduced pressure. The reddish orange residue was heated in
was achieved through the reaction between DHI and dopamine boiling deionized water for 30 min and allowed to cool to room
during the copolymerization without the need of additional post- temperature afterwards. The resulting dull yellow product was
polymerization modifications. The integrated DHI still has the collected with a yield of 74% after drying. d (300 MHz; DMSO-d ;
H
6
catechol group available to perform binding via conventional Me Si) 7.03 (1 H, s, Ar H), 7.58 (1 H, s, Ar H), 8.72 (1 H, s, CH). dC
4
4
7–49
conjugation as reported in literatures.
However, ultimately, (75 MHz; DMSO-d ; Me Si) 113.8 (C-4), 114.9 (C-9), 119.2 (C-7), 122.8
6 4
the additional coordination sites are integrated to prepare novel (C-8), 145.6 (C-6), 150.4 (C-5), 159.9 (C-3).
materials for biomedical applications which can bind and deliver
Preparation of coated silica particles and polymeric capsules
metal complexes which have a high affinity to nitrogen-based
ligands and use as therapeutics.
50–55
For instance, copper(II) com- Polymer coating was performed on silica particle templates
4
3
plexes prepared from the radioisotope copper-64 have been studied according to the procedure adapted from Peterson et al.,
62
63
extensively as theranostics for targeted radiotherapy of cancer and Postma et al., and Yu et al. with some modifications. A buffer
56–59
positron emission tomography imaging.
To this end, we used solution of pH 9–10 was prepared by dissolving tris(hydroxymethyl)-
copper(II) sulfate as a readily accessible and affordable model to aminomethane (TRIS, 242.3 mg, 2.00 mmol) in deionized water
demonstrate the enhanced binding ability over PDA with one of (120 mL). Silica nanoparticles (500 mg) were dispersed in the
the DHI/dopamine copolymers in the form of stable polymeric buffer solution by sonication. DHI (75.1 mg, 0.50 mmol) was first
capsules. We were also able to demonstrate that the incorporation dissolved in ethanol (95%, 80 mL) and added to the suspension,
of DHI has negligible impact on the cytotoxicity of PDA.
followed by the addition of dopamine hydrochloride (284.5 mg,
.50 mmol) to obtain a reaction mixture with a pH of about 8.5.
1
The mixture was stirred at room temperature for 24 h. Upon
completion, the copolmyer-coated particles were separated from
the other free-floating materials by centrifuging at 3000 rpm for
Experimental
Materials
5
min. The supernatant was decanted and the particles were
Boron tribromide (BBr3) solution, dopamine hydrochloride, redispersed in fresh water. The procedure was repeated until the
tris(hydroxymethyl)aminomethane (TRIS, Z99.8%), Dulbecco’s supernatant became clear and colorless. The particles were then
phosphate buffered saline (DPBS), and hydrofluoric acid (48%) collected by Millipore filtration and rinsed with several portions
s
were purchased from Sigma Aldrich. Aerosil OX 50 fumed of deionized water. The collected particles were dried at 40 1C in
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vacuum oven overnight. Coated particles were also synthesized Characterization
by polymerizing only dopamine (379.3 mg, 2.00 mmol) in the
presence of silica templates in aqueous TRIS buffer. Similar
procedures were performed to synthesize the copolymers of DHI
and dopamine in 1 : 1 and 1 : 3 molar ratios (1: 1-copolymer and
1
13
H, C and 2D nuclear magnetic resonance (NMR) spectro-
scopies were performed on a Bruker Avance III 300 MHz
spectrometer equipped with a SampleXpress automatic sample
changer and BBFO z-gradient probe. C solid-state (CP-MAS)
13
1
: 3-copolymer respectively, see ESI†).
spectroscopy was performed on a Bruker Avance III 300 Solid
State spectrometer equipped with a 4 mm 1H/X broadband CP-
MAS (cross polarization-magic angle spinning) probe. Ultraviolet-
visible (UV-Vis) spectroscopy was performed on a Varian Cary 300
UV-Visible Spectrophotometer using deionized water as the solvent/
dispersant unless stated otherwise. Attenuated total reflectance
Fourier transform infrared (ATR-FTIR) spectroscopy was performed
on a Bruker IFS 66/S single-beam spectrometer. Gel permeation
chromatography (GPC) was performed by a unit purchased from
Shimadzu Scientific Instruments, which included a SIL-10AD VP
To obtain capsules of the copolymer and PDA, the coated
particles were suspended in diluted hydrofluoric acid (5% v/v).
The mixture was stirred at room temperature overnight to ensure
the silica core was completely etched away. The resulting polymer
capsules were collected by Millipore filtration, followed by rinsing
with large portions of deionized water until the filtrate was neutral.
The collected capsules were purified by redispersing in deionized
water and dialyzed against DPBS solution (once per 24 h for two
days), and then against deionized water (once per 24 h for one day).
The purified capsules were retrieved by lyophilization.
À1
auto-sampler, a LC-20AT pump (set at 1 mL min ), a CTO-10A VP
column oven (set at 50 1C), and a RID-10A refractive index (RI)
Monitoring of DHI self-polymerization
5
4
detector. The chromatography columns (Phenogel – 10 , 10
A small volume of the reaction mixture (16 mL) containing
3
and 10 Å pore size) were purchased from Phenomenex. N,N-
Dimethylacetamide (DMAc) was employed as the mobile phase.
Dynamic light scattering (DLS) and zeta potential measurements
were performed on a Malvern Zetasizer Nano using deionized
water as the dispersant unless specified. Thermogravimetric
analysis (TGA) was performed using a Q5000 (V3.15 Build 263)
instrument purchased from TA Instruments. Transmission electron
microscopy (TEM) images were acquired with a JEOL 1400 transmis-
sion electron microscope. Scanning electron microscopy (SEM)
images were acquired with a Nova NanoSEM 230 filed-emission
scanning electron microscope. Energy dispersive X-ray spectroscopy
1
0 mM of DHI and TRIS, respectively, was sampled at the
designated time (1, 6, 10, 14, 24, 28, 48, 66.5 and 88 h). The
aliquot was then transfer to a quartz cuvette and diluted with
3
prepared sample was measured between 700 and 200 nm with
a UV-Vis spectrophotometer using blank water/ethanol mixture
for baseline correction prior to each measurement.
: 2 water/ethanol mixture (3 mL). The absorbance of the
Cell growth inhibition study of polymeric capsules
The buffer (PBS, Sigma D5652) and medium (DMEM, Sigma
D5523) used have an initial pH of 7.4, but the change in pH was
not monitored over the duration of the study. The cell culture
medium was prepared with DMEM solution which contained
fetal bovine serum (10%, Moregate Australia New Zealand) and
penicillin–streptomycin (1%, Sigma 4458). The polymeric capsule
samples were analyzed using a growth inhibition assay with murine
(EDS) was performed using a FEI Tecnai G2 20 transmission electron
microscope. Laser scanning confocal microscopy (LSCM) was
performed on a Zeiss LSM780 confocal microscope.
Results and discussion
4
dermal fibroblasts (L929) (ISO 10993-5 procedure). 5 Â 10 fibro-
À1
blasts mL were seeded onto 35 mm diameter tissue culture dishes. The synthesis of DHI has rarely been reported in the literature.
After 24 h of incubation at 37 1C in a 5%-CO
2
humidified atmo- As a result, we developed a straightforward, three-step reaction
sphere, the media was discarded and 1 mL of sample solution was sequence (Scheme 2) to obtain this essential monomer with an
added to the cells. The sample solution was prepared by first adequate yield and high purity (see NMR spectra from Fig. S1
À1
extracting 4 mg of polymeric capsule sample mL of media, or and S2, ESI†). The molecular structure of DHI has been studied
media alone (positive control) for 48 h at 37 1C. The extracted through 2D NMR (Fig. S3, ESI†) inorder to identify the corre-
solution, 1 mL, was added to 3 mL of media, sterile filtered and 1 mL sponding sites – C-3, 4 and 7 – which would potentially be
of this extract solution was added to the cells. Negative controls were reactive during the self-polymerization. In contrast to DHIn, the
also prepared with 4%, 5%, or 7.5% ethanol (EtOH) in cell culture 2-position of DHI is substituted with a tertiary amine. While C-3
media. Following an additional 48 h of incubation, the cells were may still be available for reactions, its ability for cross-linking is
trypsinized, and counted with a cell viability analyzer (Vi-cell XR,
Beckman Coulter).
Copper(II) binding trial on coated particles
The coated particles (8.00 mg) were dispersed in a solution of
copper(II) sulfate pentahydrate (100 mL, 10 mM, pH 5) and
stirred overnight. The treated particles were collected by Millipore
filtration and rinsed with copious amounts of deionized water until
Scheme 2 Synthesis of 5,6-dihydroxy-1H-indazole: (a) bromine, chloro-
form, reflux at 60 1C overnight; (b) hydrazine monohydrate, 1,4-dioxane,
the filtrate was clear and colorless. The collected particles were
dried under vacuum.
reflux at 80 1C overnight; (c) boron tribromide, dichloromethane, room
temperature for 4 hours.
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significantly lower because of the resulting steric hindrance as
The temporal change in UV-Vis absorbance for the reaction
the self-polymerization proceeds. The consequence of this struc- mixture was monitored over an 88 hour period. As illustrated by
tural difference will be elaborated more in later discussion. Fig. 1b, the spectra exhibited significant changes at wavelengths
Despite the seemingly comparable hydrophilic nature to below 450 nm. For instance, the dominant absorption peak at
DHIn as deduced from the molecular structure, the DHI monomer 366 nm displayed a slight blue-shift and reduction in intensity
exhibits much lower water-solubility. As a result, a solvent mixture of throughout the course of the polymerization. This absorption peak
water and ethanol in optimized ratio (3 : 2 v/v) was used as the is speculated to originate from changes in the aromaticity of the
reaction medium. The use of ethanol does not only aid the dissolu- pyrazole functionality of DHI due to the catechol group. This
tion of DHI, but also retains the ‘‘green’’ motif of the mussel-inspired influence could be due to both polymerization leading to aromaticity
chemistry. It is notable that the use of water/ethanol mixture did not changes, or quinone formation from both monomers and polymers.
affect the self-polymerization of dopamine. DHI self-polymerized at The observed linear decrease in intensity at 366 nm (Fig. 2a), there-
ambient conditions and showed a progressive change in color from fore, indicates that these changes in aromaticity are occurring
amber yellow to dark brown as the homopolymer – PDHI – was throughout the entire 88 hour period.
formed (Fig. 1a). Similar phenomenon was observed during the
preparation of the 1 : 1- and 1 : 3-copolymer (Fig. S4, ESI†).
In contrast to the peak at 366 nm, the spectral changes from
200–300 nm are characteristic for the formation of materials
similar to melanin, which in turn hint on the structure of
6
4
PDHI. Gradual increase in absorbance is observed at 284 nm
Fig. 2b) and 203 nm (Fig. 2c), respectively. Both trends approached
(
a plateau before the end of the 88 hour period, indicating that the
PDHI – a melanin analogue – had reached a stable structure, i.e.
relatively minor increases to particle size or surface coating thick-
ness. Therefore, this suggests that while the aromaticity of mono-
mers and polymers in the system is still in flux during the reaction
time, the melanin-like polymer being formed has predominantly
finished in roughly 40 h. In brief, the UV-Vis kinetics results
indicate that the self-polymerization of DHI proceeds at a signifi-
2
4
cantly slower rate than that reported for dopamine.
1
3
C solid-state NMR (CP-MAS) spectrometry was performed
to elucidate the architecture and the structural composition of
PDHI, as well as the 1 : 1- and 1 : 3-copolymers (Fig. 3). Compar-
ison was made against the spectra of the DHI monomer and
PDA. Broad clusters of peaks can be observed for the spectra of
polymeric samples, which indicate the architectural differences
with the DHI monomer. The PDHI spectrum exhibits extensive
peak-broadening at 110–130 ppm, corresponding to part of the
benzene structure of DHI. While the bridging carbons, i.e. C-8
and C-9, are inactive by nature, the broadening effect is believed
to be due to the formation of carbon–carbon bonds at C-4 and
C-7. The result suggests that PDHI consists of a p-conjugated
system as illustrated in Scheme 1, which is analogous to the
Fig. 1 Progress of the self-polymerization of DHI (a) and the spectral
change of the reaction mixture monitored by UV-Vis spectrophotometry
over 88 h (b). The inset emphasizes on the increasing absorbance at 260–
6
5
proposed structure of PDA. The absence of the C-3 peak of DHI
in all of the other spectra indicates that it is one of the reactive
3
05 nm over the course of the reaction.
Fig. 2 The change in absorbance of the self-polymerizing DHI at 366 nm (a), 284 nm (b) and 203 nm (c) monitored by UV-Vis spectrophotometry over
8 hours.
8
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Fig. 3 Comparison of the 13C solid-state NMR spectra of DHI (a), PDHI
(
b), 1 : 1-copolymer (c), 1 : 3-copolymer (d) and PDA (e).
sites during the self-polymerization. It is suspected that the
reactions at C-3 have caused the peak to shift upfield and
contributed to the broadening observed at 140–155 ppm, where
the unreactive catechol C-5 and C-6 are. Unlike DHIn, linear/
branched structures are more likely to be developed from C-3 instead
of cross-linking due to the inherent steric hindrance as depicted
previously by the molecular structure of DHI (Scheme 2).
Fig. 4 Comparisons of the UV-Vis spectrum between: DHI and PDHI (a),
dopamine and PDA (b), and the polymeric samples.
The spectra of 1 : 1- and 1 : 3-copolymer resemble that of PDA The solubility of the material aligns with the amount of DHI
due to the high degree of overlapping in the aromatic regions, that have been incorporated – the higher the DHI content the
which is attributed to the high architectural similarity between higher the solubility. The increase in solubility indicates that
PDHI and PDA. However, peaks corresponding to the aliphatic the incorporation of DHI potentially lowers the cross-linking
amine of uncyclized dopamine can be located at 20–50 ppm. density of PDA and copolymer systems by introducing more
The peak intensity increases and the signals are sharper with the mobile linear/branched features to the overall structure.
increasing dopamine content, suggesting the successful incorpora-
Gel permeation chromatography was performed on the soluble
tion of dopamine with DHI. A similar trend is observed from the fractions of the synthesized materials, whereas the analysis has
aromatic shoulder peak at 100 ppm, and also from the weak been proven inapplicable to PDA due to its insoluble nature. As
carbonyl signals of PDA at 170–190 ppm.
illustrated by Fig. 5a, the molecular weight (MW) distribution of the
13
In addition to C solid-state NMR, UV-Vis spectrophotometry synthesized materials skewed towards high-MW, with Mn
E
À1
also provides evidence to confirm the generation of new materials 20 000 g mol and a broad dispersity of Ð Z 1.48 (Table S3, ESI†),
after the self-polymerization of DHI. The spectral features become which is indicative that PDHI and the copolymers are truly poly-
more distinctive by using water as the dispersant for DHI and meric despite the presence of traces of oligomeric species in the
PDHI. The shifts in peak position are significant while a new peak samples. The high dispersity and lack of correlation between the
has emerged at 325 nm (Fig. 4a), which is analogous to the spectral DHI content and MW of the sample are results of the uncontrolled
change observed dopamine self-polymerized to PDA (Fig. 4b). nature of the self-polymerization.
Furthermore, the spectra of 1 : 1- and 1 : 3-copolymers exhibit
Being structurally similar to PDA has granted these newly
features (at 231, 281 and 318 nm) analogous to those in PDA prepared polymeric species the potential to perform surface
spectra, although being red-shifted (Fig. 4c). The shift is believed to coating in a simple, one-step, aqueous-based procedure. In the
have originated from incorporating PDHI into the structure of PDA, presence of silica particle templates, an in situ coating formed
i.e. copolymerization has taken place. Similar effects can be directly onto the particle surface as the self-polymerization pro-
observed from the ATR-FTIR spectra of the copolymers (Fig. S5, ceeded over 24 hours. However, only the 1 : 3-copolymer can form a
ESI†). However, the differences are less obvious due to the highly coating with comparable structural stability as PDA, while 1 : 1-
similar bonding constitution shared between PDHI and PDA.
copolymer and PDHI homopolymer were observed to delaminate
PDHI was found to be completely soluble in DMAc, which from the template during the cleaning process (Fig. S6, ESI†). As
suggested that the polymer does not exhibit the same degree of previously mentioned, we speculated that the DHI polymerization
cross-linking as the highly insoluble PDA. The 1 : 1- and 1 : 3- reduced cross-linking by introducing linear/branched structures
copolymers were partially soluble in DMAc, with a soluble which are more mobile. The observance of delamination and
content of 51% and 26%, respectively, as quantified from increased solubility of the generated polymer with increasing DHI
the residual material on the syringe filter (Table S2, ESI†). content would support this finding.
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proceeded (Fig. 5d). The incorporation of DHI, conversely, has
introduced an 8 hour induction period before establishing a
measurable coating with a stable on-set degradation temperature.
Since the copolymer started to degrade at lower temperatures, this
material is not as thermally stable as PDA. In short, DHI has
minimal influence on the self-polymerization kinetics of dopamine
as a comonomer even though it has been shown to exhibit a slower
polymerization rate, while it has a more significant impact on the
structural properties when incorporated into PDA by lowering the
density of the coating as well as the degree of cross-linking.
A more in-depth study of the 1 : 3-copolymer coating on silica
particles was carried out with TEM and SEM (Fig. 6a and b).
Uniform coating has been formed on the particle surface with
no observable defects. The copolymer demonstrated adequate
qualities as a surface coating. Afterwards, copolymer capsules
(1: 3, DHI-to-dopamine) were prepared by redispersing the
Fig. 5 Molecular weight distribution of PDHI, 1 : 1- and 1 : 3-copolymer
obtained by GPC (a), and kinetics of coating thickness growth (b), increase coated particles in a 5% HF solution overnight to etch the silica
of organic content (c) and change in on-set degradation temperature (d). core (Scheme 1c). As illustrated by the TEM image of the etched
particles (Fig. 6d), the solid core seen in the coated particles no
longer existed while a cavity was resulted. Similar to the coated
Since the 1 : 3-copolymer is able to form the most stable particles, the capsules did not show any major defect. Although
coating on silica templates, the application aspect of DHI has the kinetics study suggests the copolymer coating has a lower
been focused on this copolymer material. The influence of DHI density than PDA, the thicker capsule wall is believed to enhance
on the self-polymerization kinetics of dopamine was first the structural integrity and prevented the capsules from collap-
determined by monitoring changes to the 1 : 3-copolymer coating sing as indicated by SEM images (Fig. 6e).
on silica particles over time. As shown by the DLS measure-
The elemental composition of the coated particles and
ments, the coating thickness for the 1 : 3-copolymer increased capsules were analyzed by EDS. Both samples are expected to
more rapidly than that for PDA, while both traces plateaued show peaks of C, N, and O. While the Cu peak originated from
within 24 hours (Fig. 5b). In contrast, the TGA result shows that the copper grid, the Br peak is suspected to have originated
both the 1 : 3-copolymer and PDA coatings exhibited a similar from residual bromide during the monomer synthesis. In
organic content which increased at the same rate and reached a contrast to the spectrum of the particles (Fig. 6c), the absence
maximum within 24 hours (Fig. 5c). Since thickness is correlated of the Si peak and the substantial reduction of the O peak in the
to volume, the 1 : 3-copolymer is believed to have generated a spectrum of the copolymer capsules (Fig. 6f) indicates the
coating of lower density than PDA by embodying similar amount complete removal of the silica core. The TEM insets also reveal
of material within a larger volume. The lower density of the that the capsules are more transparent to the electron beam
coating is attributed to the presence of more branched structures compared to the particles, which further proves that a hollow
in DHI-incorporated polymers.
interior has been generated. The absence of silica was also
The rapid establishment of high on-set degradation tempera- confirmed by results acquired from TGA and ATR-FTIR for the
tures for the PDA kinetic samples suggests that the coating had copolymer capsules (Fig. S7, ESI†). To further elucidate the
immediately stabilized by cross-linking as the self- polymerization hollow nature of the nanocapsules generated, LSCM was
Fig. 6 Characterisations performed with TEM (a and d, scale bar = 100 nm), SEM (b and e, scale bar = 400 nm and 500 nm), and EDS (c and f) for 1 : 3-
copolymer-coated silica particles (left) and 1 : 3-copolymer capsules (right), respectively. The insets illustrate the area being irradiated by X-ray during EDS
analysis (scale bar = 500 nm).
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performed using Nile red as the fluorescent dye. Images in the
ESI† illustrate the cavity being fully fluorescent and thus
support the EDS-TEM data for hollow nanocapsule generation
(Fig. S8, ESI†).
Since occasional aggregation has been observed for both
copolymer-coated particles and copolymer capsules (Fig. S9,
ESI†) in the dry state, the zeta potential (z) of these samples was
measured and summarized in Table S4 (ESI†) to help determine
the respective colloidal stability. Briefly, the two copolymer
samples can be classified as moderately stable colloids due to
the negative z values, À35.8 Æ 0.2 mV for the coated particles
and À42.9 Æ 0.8 mV for the copolymer capsules, indicative of a
low tendency for particle aggregation. PDA-coated particles and
PDA capsules were also analyzed as a control comparison, and the z
values were found to be À32.4 Æ 0.9 mV and À8.9 Æ 1.3 mV,
respectively. Interestingly, these results suggest that PDA capsules
have the highest tendency to aggregate out of all samples. Although
aggregates would still form quite readily, the incorporation of DHI
has significantly enhanced the colloidal stability of the prepared
polymeric capsules.
Polymeric capsules based on PDA have gained attention in
the biomedical field as cargo carriers for their simple aqueous-based
6
2,63,66
preparation, promising properties, and low cytotoxicity.
The further functionalization of PDA capsules would widen their
applications, while care must be taken to not increase their
cytotoxicity. We previously found that the incorporation of 5HI at
4
3
5
0 mol% had no impact on PDA cytotoxicity, whereas a
polymerizable indazole, i.e. DHI, may elicit a different cytotoxic
response. Hence, the cytocompatibility of 1 : 3-copolymer capsules
is of immense interest for the present work. A growth inhibition
assay with murine dermal fibroblasts (L929) (ISO 10993-5
procedure) was performed to investigate the cytotoxicity of
Fig. 7 Cell growth inhibition of capsules prepared from the 1 : 3-copolymer
and PDA (a); UV-Vis results obtained from Cu(II) binding study (b).
1
: 3-copolymer capsules, and compared to PDA capsules.
peaks of the 1 : 3-copolymer have disappeared after CuSO4-
Both 1 : 3-copolymer and PDA capsules are cytocompatible treatment while a new peak has risen at about 260 nm. The
as evidenced by the minimal cell growth inhibition compared strong absorption of CuSO below 240 nm is also observed from
4
to extract media (Fig. 7a). Although the copolymer inhibited the the spectrum of the treated 1 : 3-copolymer (Fig. S10, ESI†). The
growth of L929 cells to a greater extent than PDA, the level is observed spectral changes were primarily attributed to the
still far below the 30% inhibition threshold specified in the ISO integrated DHI or, to be specific, the presence of pyrazole
4
0–42
1
0993-5 procedure. Therefore, the copolymer capsules are not moieties as ligands.
It should be noted that binding of
2
+
considered as cytotoxic. All ethanol negative controls show Cu is still achievable through the catechol groups present in
significantly higher levels of growth inhibition. The incorporation the 1 : 3-copolymer even though the change may not be detect-
of DHI without inducing significant adverse effect on the cytotoxicity able. Therefore the UV-Vis results of the copolymer samples
level of PDA is significantly beneficial to the potential application of suggest that further binding of Cu can be achieved through
the copolymer capsules as therapeutics carriers.
the pyrazole rings. More binding studies with different metals
2
+
To demonstrate the potential of these DHI integrated capsules will be required to gain insights of the loading efficiency and
to bind metal-complexed therapeutics, the binding of a copper- site selectivity.
based solution was probed as a model system. We performed a
preliminary binding trial by dispersing the 1 : 3-copolymer and PDA
samples in 10 mM copper(II) sulfate (CuSO
ca. 18 h). It is notable that no change in color or pH (i.e. stayed
blue and at pH 5) has been observed after binding for both set-ups. To conclude, fabrication of novel functional materials based on
4
) solution overnight
Conclusions
(
2
+
UV-Vis analyses of the cleaned and dried samples indicate that Cu
mussel-inspired chemistry do not always require a monomer
67
has bound to the pyrazole moieties of 1 : 3-copolymer. As illustrated derived from dopamine. Heterocyclic catechol derivatives are
in Fig. 7b, there are no observable changes to the PDA samples after potential monomer candidates provided that the selected com-
being treated by CuSO
because of the catechol groups,
4
although PDA is a good copper(II) absorbant pound can undergo self-polymerization in similar aqueous-
68,69
In contrast, the broad absorption based, oxidizing conditions. In our case, the compound DHI
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Journal of Materials Chemistry B
was chosen so as to incorporate a potential metal and drug
binding site into the coating. Further investigation is yet to be
undergone to identify the tailored applications for PDHI and
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: 1-copolymer based on their unique solubility, architecture, and
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uptake and release behavior.
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1
1
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K.W.F. and J.J.R. performed experimental work and analyzed
experimental results. K.W.F., J.J.R., and A.M.G. wrote the manu-
script. P.J.M., M.H.S., and A.M.G. conceived and designed the
project.
2013, 4, 2696–2702.
1
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1
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Conflict of interest
5 R. Liu, Y. Guo, G. Odusote, F. Qu and R. D. Priestley, ACS
Appl. Mater. Interfaces, 2013, 5, 9167–9171.
6 X. Chen, Y. Yan, M. M u¨ llner, M. P. van Koeverden, K. F. Noi,
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7 J.-H. Lin, C.-J. Yu, Y.-C. Yang and W.-L. Tseng, Phys. Chem.
Chem. Phys., 2015, 17, 15124–15130.
8 X. Zhang, S. Wang, L. Xu, L. Feng, Y. Ji, L. Tao, S. Li and
Y. Wei, Nanoscale, 2012, 4, 5581–5584.
9 Y. Liu, K. Ai, J. Liu, M. Deng, Y. He and L. Lu, Adv. Mater.,
The authors declare no competing financial interests.
Acknowledgements
We thank Dr James M. Hook and Dr Aditya Rawal for aid in
performing the solid state NMR experiments and helpful dis-
cussions of the results. The equipment in the NMR Facility at
the Mark Wainwright Analytical Centre is supported by the
Australian Research Council through Linkage Infrastructure,
Equipment and Facilities Funding Scheme (LE0989541 and
LE120100027) and the UNSW Major Research Equipment and
Infrastructure Initiative (MREII). We acknowledge the support
of the Electron Microscopy Unit (EMU) at Mark Wainwright
Analytical Centre (UNSW, Australia), especially to Ms Katie
Levick for performing EDS analysis. We thank Mr Yan Jin for
acquiring the SEM images of the samples. We also thank Dr
Hongxu Lu for acquiring the LSCM images of the dye-loaded
copolymer capsules. K.W.F. would like to acknowledge the
support of the Australian Government for scholarship funding
in the form of an Australian Postgraduate Award and a UNSW
Engineering Research Award. J.J.R. would like to acknowledge
the support of a Whitaker International Scholarship Grant.
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