Boronate ester cross-linked PVA hydrogels for the capture and H2O2-mediated release of active fluorophores

Article abstract of DOI:10.1039/d0cc01904f

A new set of PVA hydrogels were formed using the boronate ester fluorescent probe PF1 and the novel boronate fluorescent probe PT1 as the covalent crosslinkers. Treatment with aqueous H₂O₂ allowed triggered release of the fluorescent dye accompanied by complete dissolution of the hydrogel.

Full text of DOI:10.1039/d0cc01904f

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DOI: 10.1039/D0CC01904F
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Boronate ester cross-linked PVA hydrogels for the capture and
H2O2-mediated release of active fluorophores
George T. Williams,a,c† Adam C. Sedgwick,b,† Sajal Sen,b† Lauren Gwynne,a Jordan E. Gardiner,a James
T. Brewster II,b Jennifer R. Hiscock,c* Tony D. James,*,a A. Toby A. Jenkins,*,a and Jonathan L.
Sessler*b
Received 00th June 2019,
Accepted 00th Month 20xx
DOI: 10.1039/x0xx00000x
A new set of PVA hydrogels were formed using the boronate ester
fluorescent probe, PF1 and the novel boronate fluorescent probe
PT1 as the covalent crosslinkers. Treatment with aqueous H2O2
allowed triggered release of the fluorescent dye accompanied by
complete dissolution of the hydrogel.
Functional hydrogels have generated widespread interest as
so-called intelligent devices wherein a specific stimulus can
yield a macroscopic change to the self-supporting material.1, 2
Such constructs offer promise in the area of drug delivery and
design of “smart” wound dressings.3-6 In addition, these
functional hydrogels have demonstrated great potential as
fluorescent probes for live cell imaging, disease diagnosis and
Scheme 1 - Cartoon representation illustrating the boronate pro-
fluorophore encapsulated within a PVA hydrogel being activated by
H2O2 to release the active fluorophore.
Boronic acid and boronate esters have found widespread
application in material-based applications, in part because of
their propensity to bind reversibly with 1,2-and 1,3-diols.9-19
Such chemistry has been demonstrated inter alia using
commercially available polyvinyl alcohol (PVA) and diboronic
acid crosslinkers to afford functional PVA-boronate
hydrogels.20-25 Boronic acids and boronate esters are well-
known to undergo hydrogen peroxide (H2O2)-mediated
oxidative transformations to afford their corresponding phenol
functionalites.26-28 We envisaged that the use of bis-boronate-
based pro-molecules as cross-linkers would afford a H2O2-
responsive hydrogel platform that would allow the controlled
and localised release of an active molecule, such as a
fluorophore (Scheme 1). It is important to note the boronate
functionality is commonly used to mask active therapeutics.29, 30
Currently, there is considerable interest in functionalized
hydrogels wherein a specific stimulus can yield a macroscopic
change to the self-supporting material, including for the
stimulus-based release of specific payloads.31, 32,33, 34 However,
new approaches to achieving such overarching objectives are
still needed.
sensing applications with the controlled release of
a
fluorophore.7 These constructs have utilised non-covalent
interactions such as aromatic–aromatic, hydrogen bonding, and
hydrophobic interactions. Unfortunately, these interactions can
result in the unwanted leaching of the active molecule from the
hydrogel matrix. Next generation systems comprised of a pro-
molecule backbone covalently linked to the hydrogel may
address these issues by providing a higher local dose and
sustained/controlled release of the bioactive molecule.8 Here,
we demonstrate a new set of controlled release materials
wherein hydrogen peroxide (H2O2) is used as a stimulus to
release fluorophores from polyvinyl alcohol (PVA) boronate
hydrogels.
a. Department of Chemistry, University of Bath, Bath, BA2 7AY, UK. Email:
t.d.james@bath.ac.uk, a.t.a.jenkins@bath.ac.uk
b. Department of Chemistry. University of Texas at Austin, 105 E 24th street A5300,
Austin, TX 78712-1224, United States. Email: sessler@cm.utexas.edu
c. School of Physical Sciences, University of Kent, Canterbury, CT2 7NH, U.K. Email:
J.R.Hiscock@kent.ac.uk
To address the above need, we have now prepared a new
class of H2O2-responsive PVA-boronate hydrogels. These
systems rely on covalent cross-linking provide solely by a set of
constituent H2O2-responsive boronate ester fluorescent probes,
namely the known fluorophore PF126 and the novel fluorescent
† These authors contributed equally.
Electronic Supplementary Information (ESI) available: [details of any supplementary
information available should be included here]. See DOI: 10.1039/x0xx00000x
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probe, PT1 (Figure 1). The resultant hydrogel constructs fluorescence imaging of both ONOO– and H2OV2i,ewaAlrbtieclietOnnlionet
DOI: 10.1039/D0CC01904F
Greenment (Gment) and Purplement (Pment) displayed necessarily in a species specific manner.
stability over 7 days in both aqueous solution and in the air;
Next, the Gment and Pment PVA-hydrogels were prepared
however, upon exposure to aqueous H2O2 the polymers were by mixing a solution of either PF1 or PT1 (100 mM) in
oxidised thus releasing their constituent fluorophores, dimethylsulfoxide (DMSO) with a DMSO solution of 10% PVA
fluorescein26 and thionol35 (Schemes S1 and S2). Complete (low molecular weight; purchased commercially) in a 1:1 ratio.
dissolution of the hydrogel could be effected depending on the This solution was then heated to induce gelation, followed by
specific choice of conditions as detailed below.
heating at 60 °C overnight in an oven. The resultant gels were
washed with hexanes to remove the displaced pinacol and
water to remove excess DMSO. These self-supporting gels
proved physically robust and stable in air and could be stored in
aqueous media (PBS, pH 7.4) without degradation for 7 days
until used Fig. S6-S8.
The ability of Gment or Pment-based PVA-hydrogel to
release the corresponding dye in the presence of H2O2 was then
evaluated. This was done by submerging the chosen hydrogel
(200 ± 10 mg) in aqueous solutions containing different
concentrations of H2O2. As shown in Figure 2, exposure of
Gment gels to H2O2 (0 – 1 mM) led to a dose-dependent increase
in the fluorescence emission intensity. The colorimetric nature
of Gment was then tested by placing the hydrogel (200 ± 10 mg
samples) in an aqueous solution of H2O2 (1 mL, 1 mM). A change
in colour from colourless to green ensued. Analysis of the UV-
Vis absorption revealed an increase in two absorption peaks at
450 nm and 490 nm (Fig. S9). The absorption peak at ~ 450 nm
is tentatively assigned to the release of monoboronate PF340
and the absorption peak at 490 nm corresponds to the release
of fluorescein. Based on this result, we believe that oxidation of
only one boronate linkage is required to release the fluorescent
cargo from the PVA-hydrogel system (Scheme S3).
Figure
corresponding H2O2-responsive PVA hydrogel Gment
responsive fluorescent probe PT1 and its corresponding H2O2-
responsive PVA hydrogel Pment
1
-
(a) H2O2-responsive fluorescent probe PF1 and its
.
(b) H2O2-
.
PF1 was prepared following literature procedures.26 The novel
fluorescent probe PT1 was synthesized through the
dibromination of commercially available phenothiazine (1)
using Br2 (5 equiv.) in acetic acid at room temperature, giving
the desired product in 74% yield. Subsequent Suzuki-Miyaura
borylation using potassium acetate, bis(pinacolato)diboron, and
Pd(dppf)Cl2 afforded PT1 in 43 % yield.
With PF1 and PT1 in hand, UV and fluorescence analyses were
performed. Upon exposure to aqueous H2O2 at concentrations
as low as 125 μM, PF1 exhibited a colour change from clear to
green with an increase in absorption at 490 nm and an increase
in fluorescence emission at 520 nm, which corresponded to the
release of fluorescein (Fig. S1 and S2). Whereas, exposure of
PT1 to H2O2 in an analogous manner led to a colour change from
clear to purple and a concomitant increase in the absorption
intensity at 595 nm and an increase in fluorescence emission at
610 nm. These optical changes reflected the release of free
thionol as confirmed by high resolution mass spectrometry, Fig.
S3-S5. It is important to note that in this work, we have focused
on the use of these boronate-based hydrogels as materials
whose controlled release may be triggered by H2O2. However,
previous reports have demonstrated the greater reactivity of
Figure 2 – Fluorescence spectra of the supernatant of Gment-based
PVA- hydrogels exposed to various concentrations of H2O2 (0 – 1 mM) in
PBS, pH 7.4. Measurements were taken after 5 min at 25 °C. λex = 472
(bandwith: 16 nm) on a BMG Labtech CLARIOstar® plate reader.
As shown in Figure 3, Pment PVA-hydrogels exposed to various
concentrations of H2O2 (0 – 1 mM) also led to a dose-dependent
increase in the fluorescence intensity at the emission maximum
of 610 nm. The colorimetric nature of Pment was then tested
by placing the hydrogel (200 ± 10 mg samples) in an aqueous
solution of H2O2 (1 mL, 1 mM). A readily discernible change in
colour was observed from colourless to purple with an increase
in the absorption intensity at 595 nm (See ESI - Figs. S9 - S11). In
comparison to one another, Gment was found to be more
boronate-based fluorescent probes towards peroxynitrite
36-39
(ONOO–).16,
Therefore, it is likely that if used in cellular
applications, both PF1 and PT1 could have a role to play in the
2 | J. Name., 2012, 00, 1-3
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sensitive to H2O2 than Pment (Figs. S12 and S13). This finding is synthesis of the previously reported H2O2-responsive
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reflected in Gment having a lower Limit of Detection ((LoD) - fluorescent probe PF1. Both PT1 and PF1 wDOeIr:e10s.u10c3c9e/sDs0fCuClly01u9s0e4dF
Gment = 0.12 mM, LoD Pment = 0.33 mM). However, it is as diboronic acid crosslinkers to form air and aqueous stable
important to note, these calculated LoD values are dependent PVA-based hydrogels. Exposure of these initially colourless and
upon incubation times.
non-fluorescent systems to aqueous solutions of H2O2 allowed
for the controlled release and activation of the encapsulated
fluorophore. We believe these systems represent a masking and
delivery strategy that has the potential to achieve the controlled
and localised release of boronic acid-based sensors and pro-
drugs.29
Acknowledgments
The authors would like to thank the EPSRC for grant EP/
R003556/1. G.T.W. would like to thank the EPSRC and Public
Health England. G.T.W. and J.R.H. would like to thank the global
challenges doctoral centre at the University of Kent for funding.
A.C.S. and A.T.A.J. wish to thank the EPSRC for funding on smart-
wound plasma – EP/R003939/1. This work was supported in
part by grant MR/N0137941/1 for the GW4 BIOMED DTP,
awarded to the Universities of Bath, Bristol, Cardiff and Exeter
from the Medical Research Council (MRC)/UKRI. T.D.J. wishes to
thank the Royal Society for a Wolfson Research Merit Award.
The work in Austin was supported by the National Institutes of
Health (R01 GM103790 to J.L.S.) and the Robert A. Welch
Foundation (F-0018 to J.L.S.). A.C.S. would also like
to acknowledge use of a Bruker AVIII HD 500 with Prodigy
liquid nitrogen cryoprobe supported by NIH grant 1 S10
OD021508. All data supporting this study are provided as
supplementary information accompanying this paper (ESI†).
Figure 3 – Fluorescence spectra of the supernatant of Pment-based
PVA- hydrogels exposed to various concentrations of H2O2 (0 – 1 mM) in
PBS, pH 7.4. Measurements were taken after 5 min at 25 °C. λex = 570
(bandwith: 16 nm) on a BMG Labtech CLARIOstar® plate reader.
Notably, subjecting the hydrogels to an aqueous solution of
H2O2 (100 mM) resulted in the complete dissolution of the
hydrogels into solution, as shown in Figure 4. Of note is that
commercially available 3% H2O2 sold for consumer use is
approximately 980 mM. The present work thus demonstrates
the potential utility of boronate-based PVA polymers as a smart
material for the masking and facile release of easy-to-visualise
fluorophores using a readily accessible trigger. Lastly, an MTT
assay with A549 cells was carried out using the Gment gel. At
concentrations up to 50 μg/mL, A549 cells displayed at least
80% viability, thus demonstrating minimal acute cytotoxicity in
this well-studied cell line (Fig. S14). We believe these findings
provide further support for the suggestion that the present
approach may prove useful in achieving the controlled delivery
Conflicts of interest
There are no conflicts to declare.
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