Biomimetic polymers responsive to a biological signaling molecule: Nitric oxide triggered reversible self-assembly of single macromolecular chains into nanoparticles

Article abstract of DOI:10.1002/anie.201403147

Novel nitric oxide (NO) responsive monomers (NAPMA and APUEMA) containing o-phenylenediamine functional groups have been polymerized to form NO-responsive macromolecular chains as truly biomimetic polymers. Upon exposure to NO - a ubiquitous cellular signaling molecule - the NAPMA- and APUEMA-labeled thermoresponsive copolymers exhibited substantial changes in solubility, clearly characterized by tuneable LCST behavior, thereby inducing self-assembly into nanoparticulate structures. Moreover, the NO-triggered self-assembly process in combination with environmentally sensitive fluorescence dyes could be employed to detect and image endogenous NO. NO problem: Polymerization of NO-responsive monomers containing o-phenylenediamine functional groups has led to the formation of NO-responsive macromolecular chains that act as truly biomimetic polymers. Exposure to NO results in the thermoresponsive copolymers undergoing self-assembly into micellar structures (see example). The NO-triggered self-assembly process with turn on fluorescence was further used to image endogenous NO.

Full text of DOI:10.1002/anie.201403147

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Chemie
DOI: 10.1002/anie.201403147
NO-Responsive Polymers
Biomimetic Polymers Responsive to a Biological Signaling Molecule:
Nitric Oxide Triggered Reversible Self-assembly of Single
Macromolecular Chains into Nanoparticles**
Jinming Hu, Michael R. Whittaker, Hien Duong, Yang Li, Cyrille Boyer,* and Thomas P. Davis*
Abstract: Novel nitric oxide (NO) responsive monomers
(NAPMA and APUEMA) containing o-phenylenediamine
functional groups have been polymerized to form NO-
responsive macromolecular chains as truly biomimetic poly-
mers. Upon exposure to NO—a ubiquitous cellular signaling
molecule—the NAPMA- and APUEMA-labeled thermores-
ponsive copolymers exhibited substantial changes in solubility,
clearly characterized by tuneable LCST behavior, thereby
inducing self-assembly into nanoparticulate structures. More-
over, the NO-triggered self-assembly process in combination
with environmentally sensitive fluorescence dyes could be
employed to detect and image endogenous NO.
have been designed to react to stimuli in diverse applications
such as artificial muscles, smart membranes, and triggered
delivery vectors.[5] The most widely published triggers used to
induce polymer responsiveness have been temperature,
pH value, ionic strength, external fields, and mechanistic
forces, inducing phase changes by manipulating the polymer–
solvent versus polymer–polymer interactions.[6] However,
harnessing biological signaling molecules to trigger respon-
siveness in synthetic polymer systems is relatively rare,
despite an imperative to design polymers to emulate natural
systems (biomimetic polymers).
Stimuli-responsive polymers that are responsive to intra-
cellular pH gradients, thiols, hydrogen peroxide (H2O2), and
enzymes[7] have been elegantly explored, targeting applica-
tions including intracellular imaging and diagnostic and drug/
gene delivery. However, studies on biomimetic polymers
responsive to intracellular signaling molecules (i.e. NO,
carbon monoxide (CO), and hydrogen sulfide (H2S)) are
rare, despite the widespread cellular presence of these
messenger molecules. In recent years, there have been some
reports describing the use of carbon dioxide (CO2) sensitive
polymers, mostly exploiting amidine and tertiary amine
functionalities, and this has become a topic of growing
international interest, as reflected by a steady increase in
publications.[8] CO2-responsive polymers presage a new field
of stimuli-responsive polymers, triggered by simple gaseous
molecules present physiologically. We therefore decided to
exploit NO concentration as a trigger for assembling macro-
molecules, and to our knowledge this is the first example
where NO is reported as a molecular trigger that induces
responsiveness in synthetic polymer chains, thereby driving or
influencing self-assembly, in a truly biomimetic process.
Published research on novel fluorescence sensors for
in vitro and in vivo sensing and also imaging of NO provided
some knowledge on potentially useful NO reactive chemis-
try.[9] Various sensing mechanisms including selective NO-
triggered reactions and NO-mediated displacements or
reduction of metal ions have all been explored.[10] In previous
studies describing the fabrication of NO-sensitive probes, the
electron-rich o-phenylenediamine group has been frequently
utilized as a fluorescence quencher moiety through a photo-
induced electron-transfer (PET) mechanism.[11] o-Phenylene-
diamine groups are reactive towards NO, producing benzo-
triazole moieties in a highly efficient and highly selective
manner in the presence of O2. Partially inspired by previous
studies, we envisioned that novel NO-responsive poly-
mer chains could be designed by exploiting highly reactive
o-phenylenediamine moieties. Herein, we demonstrate
the synthesis of NO-responsive monomers and their incorpo-
N
itric oxide (NO), known as an atmospheric pollutant and
a potential health hazard, is recognized as an endothelium-
derived relaxing factor (EDRF) and a broad-spectrum
biological signaling molecule that operates at both systemic
and specific cellular levels.[1] NO is a gaseous radical species
with a half-life of several seconds, biosynthesized in living
tissues, and functions as a ubiquitous messenger molecule in
the cardiovascular, nervous, and immune systems in animals
(and also within plant life).[2] NO exerts influence over
critically important physiological activities (i.e. as an EDRF
in blood vessels, a neurotransmitter in the central nervous
system, and a mediator in the immune system).[3] Misregula-
tion of NO is thought to be a factor in numerous human
diseases including cardiovascular disorders, gastrointestinal
distress, neurodegeneration, and hypertension.[4] In a drive to
emulate natural systems and processes, synthetic polymers
[*] Dr. J. Hu, Dr. M. R. Whittaker, Y. Li, Prof. Dr. T. P. Davis
ARC Centre of Excellence in Convergent Bio-Nano Science and
Technology, Monash Institute of Pharmaceutical Sciences
Monash University, Parkville, VIC 3052 (Australia)
Prof. Dr. T. P. Davis
Chemistry Department, University of Warwick
Coventry, ULCV4 7AL (UK)
E-mail: Thomas.P.Davis@monash.edu
Dr. H. Duong, Prof. Dr. C. Boyer
Australian Centre for NanoMedicine
School of Chemical Engineering, University of New South Wales
Sydney NSW 2052 (Australia)
E-mail: cboyer@unsw.edu.au
[**] T.P.D. and C.B. acknowledge the Australian Research Council (ARC)
for funding in the form of a Discovery grant (DP130100107) and
Centre of Excellence funding (T.P.D.). In addition, we would like to
acknowledge ARC Future Fellowship funding to C.B.
(FT120100096).
Supporting information for this article is available on the WWW
under http://dx.doi.org/10.1002/anie.201403147.
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ration into thermoresponsive copolymers, thereby provid-
ing the opportunity to delicately mediate the thermo-
responsive behavior of polymers by using an NO stimulus
(Scheme 1).
ure 1b and d). The final products, after reaction with NO,
were separated and characterized by NMR spectroscopic
analyses (see Figures S4 and S5 in the Supporting Informa-
tion), thereby providing evidence for the formation of an
amide-substituted
benzotriazole
derivative and a urea-functionalized
benzotriazole residual, respectively.
Our titration results clearly indicate
that the NAPMA and APUEMA
monomers are highly reactive with
NO under extremely mild condi-
tions (aqueous solution and room
temperature). Since the effect of
NO in biological environments is
highly concentration-dependent,[12]
the
current
highly
sensitive
NAPMA and APUEMA monomer
structures may augur promising
intracellular applications.
Next, we further investigated
the stabilities of the newly formed
products in aqueous solution by
UV/Vis spectroscopy. Interestingly,
the aqueous solution of the amide-
substituted benzotriazole exhibited
an absorption centered at 302 nm, in
accord with the titration data (see
Figure 1a). Upon extending the
incubation time, the amide-substi-
Scheme 1. Schematic representation showing the reversible regulation of the lower critical solution
temperatures (LCSTs) of NO-responsive copolymers, P(NIPAM-co-NAPMA) and P(NIPAM-co-
APUEMA), using NO as a molecular trigger.
tuted
benzotriazole
gradually
hydrolyzed, as evident by a slowly
decreasing absorption at 302 nm.
Typically, the hydrolysis of the
The NO-responsive monomers N-(2-aminophenyl) meth-
acrylamide hydrochloride (NAPMA) and 2-(3-(2-aminophe-
nyl)ureido)ethyl methacrylate hydrochloride (APUEMA)
were easily synthesized by treating equivalent amounts of o-
phenylenediamine with methacryloyl chloride or 2-isocyana-
toethyl methacrylate, respectively, (see Scheme S1a and S1b
in the Supporting Information) and the chemical structures of
NAPMA and APUEMA monomers were confirmed by NMR
spectroscopic analyses (see Figures S1–S3 in the Supporting
Information).
amide-substituted benzotriazole was complete within 2 h
(Figure 2a,b). A similar hydrolysis phenomenon was also
observed in o-phenylenediamine-containing NO probes.[11b,d]
By sharp contrast, the absorption spectra of the urea-
To investigate the NAPMA/NO and APUEMA/NO
reaction kinetics we titrated NO into aqueous solutions of
NAPMA and APUEMA monomers and monitored the
ensuing reaction by UV/Vis spectroscopy. The NAPMA and
APUEMA monomers exhibited absorptions centered at
about 288 and 286 nm, respectively (Figure 1a and c). Upon
addition of NO (0–48 mm), the absorption band of NAPMA
decreased slightly and then red-shifted to 302 nm, whereas
the absorption band of APUEMA gradually increased in
intensity, which was associated with the co-appearance of two
bands, located at about 258 nm and 295 nm (Figure 1a and c).
If we arbitrarily defined the NO detection limit as the
concentration at which a 10% change in absorbance could be
measured at 302 nm or at 258 nm, the NO detection limits
were then determined to be about 2.5 mm or 2.4 mm, respec-
tively, from the two characteristic absorption bands (Fig-
Figure 1. a,c) UV/Vis spectra and b,d) absorption intensity changes
recorded for aqueous solutions of (a,b) the NAPMA monomer and
(c,d) the APUEMA monomer (20 mm) upon addition of various NO
concentrations.
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Figure 2. a,c) UV/Vis spectra and b,d) changes in the absorption
intensity of aqueous solutions of (a and b) the NAPMA and (c,d)
APUEMA monomer after reaction with NO.
functionalized benzotriazole derivative remained unchanged
after a long time (e.g. 24 h), thus indicating that the urea-
functionalized benzotriazole is relatively stable in aqueous
solution (Figure 2c,d).
The hydrolysis products of the NAPMA monomer
following reaction with NO revealed the coexistence of
benzotriazole and methacrylic acid (MAA) groups, as evident
by 1H NMR spectroscopic analysis (see Figure S6 in the
Supporting Information). After purification, the final hydrol-
ysis product was identified by NMR spectroscopy, thereby
confirming the formation of benzotriazole (see Figure S7 in
the Supporting Information). The chemical conversion of the
NAPMA monomer in the presence of NO was further
characterized by FTIR spectroscopy (see Figure S8 in the
Supporting Information), which showed that, after hydrolysis,
absorption band corresponding to the amide group (at
1670 cmꢀ1) disappeared. Moreover, the absorption spectrum
of the final hydrolysis product is relatively similar to that of
the benzotriazole (see Figure S9 in the Supporting Informa-
tion), further confirming our proposed mechanism
(Scheme 2a).
From our preliminary results, we thus ascertained that the
NAPMA monomer could react with NO under extremely
mild conditions to yield an amide-substituted benzotriazole
intermediate that is sensitive to hydrolysis in an aqueous
solution to form benzotriazole and residual carboxyl groups
(Scheme 2a). The APUEMA monomer could also respond to
NO under the same conditions, as confirmed by NMR and
FTIR spectroscopic analyses (see Figures S3 and S10 in the
Supporting Information), thereby resulting in a relatively
stable urea-functionalized benzotriazole unsusceptible to
hydrolysis in aqueous solution (Scheme 2b). We therefore
envisaged that NO-triggered processes could be exploited to
induce unique compositional transitions in macromolecular
chains (incorporating NAPMA and APUEMA monomer
units) for potentially manipulating polymer phase transitions
and inducing self-assembly.
Scheme 2. Schematic illustration of the proposed NO-triggered mech-
anisms of copolymers bearing NAPMA and APUEMA moieties.
Consequently, we synthesized well-defined NAPMA-
containing and APUEMA-labeled thermoresponsive copoly-
mers,
P(NIPAM-co-NAPMA)
and
P(NIPAM-co-
APUEMA), through reversible addition-fragmentation
chain transfer (RAFT) polymerization. The copolymers
exhibited symmetrical molecular weight distributions with
relatively low polydispersities (PDI < 1.1), as evident by size-
exclusion chromatography (SEC) analyses (see Figure S11 in
the Supporting Information). This result demonstrated that
the NAPMA and APUEMA monomers can be successfully
copolymerized using RAFT polymerization. The degrees of
polymerization of the copolymers and molar contents of
NAPMA and APUEMA were determined by 1H NMR
spectroscopy (see Figures S12a and S13a in the Supporting
Information; the measured structural parameters of the
thermoresponsive copolymers are summarized in Table S1).
Provided that there are free amine groups in the mono-
mers, they can influence the thermoresponsive properties of
the copolymers. Subsequent pH-titration experiments with
the NAPMA and APUEMA monomers gave pKa values of
3.46 and 3.36, respectively (see Figure S14 in the Supporting
Information), thus confirming that the amino groups are in
a neutral state at pH 7.0.
It is well-documented that the PNIPAM homopolymer
has a lower critical solution temperature (LCST) at around
328C in aqueous solution.[13] The P(NIPAM-co-NAPMA) and
P(NIPAM-co-APUEMA) copolymers also exhibited LCST
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behaviors in aqueous solution, as evident by dynamic light
scattering (DLS) analyses. Typically, the copolymers formed
aggregates with intensity-average hydrodynamic diameters
hDhi between 600 and 800 nm at 408C without NO (see
Table S1 in the Supporting Information). Temperature-de-
pendent turbidity measurements revealed that the LCST
values of the P(NIPAM-co-NAPMA) and P(NIPAM-co-
APUEMA) copolymers were 348C and 288C for copolymers
with about 5 mol% NAPMA and APUEMA monomer,
respectively, thus showing that the NAPMA and APUEMA
groups have a detectable impact on of the overall phase
transition behavior of the NIPAM-rich chains (see Table S1
and Figure S15 in the Supporting Information).
the P(NIPAM-co-APUEMA) copolymers, in contrast,
decreased upon the gradual addition of NO (see Figure S15
in the Supporting Information).
The P(NIPAM-co-APUEMA) copolymer could react
with NO thereby decreasing the LCST and presumably
resulting in the formation of aggregates. The sizes of the
P(NIPAM-co-APUEMA) copolymers, after reaction with
NO, were found by DLS to have an intensity-averaged
hydrodynamic diameter hDhi of about 37.8 nm at 208C
(Figure 3a), while TEM showed the formation of spherical
micelles (Figure 3b). These results suggest that the self-
assembly of the P(NIPAM-co-APUEMA) copolymer could
be efficiently regulated by exposure to NO. It is intriguing
that we may be able to manipulate the self-assembly behavior
of the copolymers by using a relatively low NO concentration
that falls in the biological/physiological concentration range.
Subsequently, the self-assembly behavior of P(NIPAM-co-
APUEMA) copolymer in the presence of a low NO concen-
tration was examined. To eliminate any possible side reac-
tions between the RAFT terminal group and NO, which
would potentially consume NO (see Figure S16 in the
Supporting Information), the RAFT terminal group was
removed in the presence of an excess of azoisobutyronitrile
(AIBN). The P(NIPAM-co-APUEMA) copolymer exhibited
an extremely low scattering intensity and small diameter (i.e.
5.9 nm) at 208C, which suggests that the copolymer was in
a unimer state without NO (Figure 3c,d), in line with the
temperature-dependent changes in turbidity (see Figure S15
in the Supporting Information). However, aggregates with
prominently increased scattering intensities and diameters
were observed upon gradual addition of NO. Specifically, in
the presence of 0–40 mm NO, the diameters increased from
5.9 nm (unimers) to 38 nm (micelles). Meanwhile, the corre-
sponding scattering intensity exhibited a cumulative 10.4-fold
Subsequently, we explored the thermoresponsive behav-
ior of the copolymers following addition of NO. The final
copolymers, after NO treatment, were characterized by SEC
measurements (see Figure S11 in the Supporting Informa-
1
tion) and H NMR spectroscopy (see Figures S12b and S13b
in the Supporting Information). From the SEC traces, we
discerned that after reaction with NO, the P(NIPAM-co-
NAPMA) copolymers exhibited slightly decreased elution
times while P(NIPAM-co-APUEMA) copolymers had
slightly increased elution times, which is indicative of
compositional changes (see Figure S11 in the Supporting
Information). A successful reaction of NAPMA with NO was
confirmed by the absence of the NMR signals between 7–
8 ppm assigned to the phenyl rings of the NAPMA moieties
(see Figure S12b in the Supporting Information). Although
the P(NIPAM-co-APUEMA) copolymers do not dissolve in
D2O at room temperature after reaction with NO, the
1H NMR spectrum in [D6]DMSO revealed that the protons
ascribed to the phenyl ring of APUEMA moieties were
shifted downfield, in accord with the monomer transition
after reaction with NO (see Figure S3 in the Supporting
Information). Our results confirmed that the copolymeriza-
tion of NAPMA and APUEMA monomers with NIPAM did
not compromise (or affect) the NO-responsive behavior.
The mechanisms of the reaction of the NAPMA and
APUEMA moieties with NO are given in Scheme 2, where
thermoresponsive P(NIPAM-co-NAPMA) copolymers trans-
form to P(NIPAM-co-MAA) copolymers following treatment
with NO, while the P(NIPAM-co-APUEMA) copolymers
form urea-functionalized benzotriazole moieties with long-
term stability in aqueous solution. The measured LCST of
P(NIPAM-co-NAPMA) copolymers with 5 mol% NAPMA,
after reaction with NO, was determined to be 388C at pH 7,
while that of P(NIPAM-co-APUEMA) copolymers with
5 mol% APUEMA drops to 7.58C. The increased LCST
values of P(NIPAM-co-NAPMA) are in accord with the
expected reaction mechanism following formation of nega-
tively charged carboxyl groups at pH 7.0, which results in
increased LCST values (Scheme 2a). We speculate that the
phenylamine moieties of the APUEMA monomer units
transform to hydrophobic urea-derived benzotriazole motifs,
thereby leading to decreased LCST values (Scheme 2b).
Moreover, we find that the LCST changes of the copolymers
are highly dependent on the NO concentration. Typically, the
LCST values of P(NIPAM-co-NAPMA) gradually increased
with increasing NO concentrations, while the LCST values for
Figure 3. a) Typical DLS profile and b) transition electron microscopy
(TEM) image of an aqueous solution (0.05 glꢀ1) of P(NIPAM-co-
APUEMA) ([APUEMA]=20 mm) after treatment with an excess of NO
at 208C. c) Scattering intensity and diameter changes in an aqueous
solution (0.05 glꢀ1) of P(NIPAM-co-APUEMA) upon addition of vari-
ous amounts of NO at 208C. d) Changes in the size of P(NIPAM-co-
APUEMA) in aqueous solution (0.05 glꢀ1) in the presence of 0, 2.5,
and 40 mm NO.
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increase (Figure 3c). Intriguingly, the minimally detectable
size change by DLS was determined to be approximately
2.5 mm NO, which resulted in the formation of micellar
aggregates with a diameter 18 nm (Figure 3d) and implying
the copolymer could also be employed to detect NO by
inducing assembly.
examined the temperature-dependent fluorescence changes.
Upon gradual addition of NO, the fluorescence intensity
underwent a 7.1-fold increase at 378C at the same NO
concentration (see Figure S18 in the Supporting Informa-
tion). The further increased fluorescence, compared to the
fluorescence at 208C, could be ascribed to the thermoinduced
collapse of NIPAM chains, thus leading to a further decreased
environmental polarity that improved the quantum yield of
NBD. This result is significant, as it indicates that NO-
responsive copolymers could be employed as temperature
sensors.[6b,14]
Finally, the performance of the NO-responsive copolymer
in living cells was assessed. The P(NIPAM-co-APUEMA-co-
NBD) copolymer was nontoxic even at concentrations of
1 mgmLꢀ1, as confirmed by assessment of the in vitro
cytotoxicity against the MRC-5 cell line (see Figure S19 in
the Supporting Information). After internalization by MRC-
5, it is intriguing that the initially quenched NBD fluorescence
could now be observed. This enhanced fluorescence is likely
due to the scavenging of APUEMA moieties by endogenous
NO, thereby resulting in the detection of fluorescence inside
the MRC-5 cells (Figure 4).
In conclusion, novel NO-responsive monomers, NAPMA
and APUEMA bearing o-phenylenediamine groups, were
synthesized and found to react effectively with NO under
extremely mild conditions to generate amide-substituted
benzotriazole intermediates and urea-functionalized benzo-
triazole derivatives, respectively. Moreover, the amide-sub-
stituted benzotriazole intermediate could be spontaneously
hydrolyzed in aqueous solution to yield carboxyl moieties
with the liberation of a benzotriazole motif, while the
resultant urea-functionalized benzotriazole derivates proved
relatively stable. The LCST values of the thermoresponsive
copolymers P(NIPAM-co-NAPMA) and P(NIPAM-co-
APUEMA) could be significantly regulated by exposure to
NO stimuli, with drastic increases and decreases in the LCST
value, respectively. The self-assembly of P(NIPAM-co-
APUEMA) could be delicately regulated at extremely low
NO concentrations by taking advantage of the NO-triggered
formation of a hydrophobic motif. Our preliminary intra-
cellular results revealed that the NO-responsive copolymer is
noncytotoxic and could respond to endogenous NO concen-
trations through fluorescence changes. This study, for the first
time, demonstrates the manipulation of thermoresponsive
copolymers using a biological signal molecule (NO) to induce
significant LCST changes, which leads to self-assembly and
fluorescence changes. Further studies of structural parame-
ters that control the size and shape of self-assembled
aggregates and the exploration of potential applications,
such as NO-triggered release and in vivo imaging, are
currently underway.
To further confirm the NO-triggered self-assembly pro-
cess, we subsequently introduced a polarity-sensitive fluores-
cent dye (4-(2-methylacryloyloxyethylamino)-7-nitro-2,1,3-
benzoxadiazole, NBD) to the P(NIPAM-co-APUEMA)
copolymers to afford P(NIPAM-co-APUEMA-co-NBD).
The fluorescence of the P(NIPAM-co-APUEMA-co-NBD)
copolymer was very weak at 208C, which could be ascribed to
two synergistic effects: 1) the NBD dye being located in
a hydrophilic environment as the copolymer was in a unimer
state; 2) the deprotonated APUEMA poses as a fluorescence
quencher through a PET mechanism. In contrast, the
copolymer exhibited a drastic increase in fluorescence upon
addition of NO, leveling off in the presence of 30 mm NO, but
exhibiting a cumulative 5.2-fold increase. This was accom-
panied by macroscopic turbidity (see inset in Figure 4a) and
scattering intensity changes (see Figure S17 in the Supporting
Information), which suggests the formation of aggregates. In
contrast, the fluorescence of P(NIPAM-co-NBD) without
APUEMA moieties displayed negligible changes at the same
NO concentration, thereby indicating an inert fluorescence
property of NBD against NO. The above results confirmed
that the NO-triggered self-assembly yielded decreased envi-
ronmental polarity, thereby resulting in an enhanced fluores-
cence of the NBD moieties (Figure 4a,b).[6b,14]
Considering the temperature-responsive nature of the
P(NIPAM-co-APUEMA-co-NBD) copolymer, we further
Figure 4. Changes in the fluorescence intensity (lex =470 nm,
lem =530 nm) of aqueous solutions (0.05 glꢀ1) of a) P(NIPAM-co-
APUEMA-co-NBD) and b) P(NIPAM-co-NBD) at pH 7.0 with various
amounts of NO at 208C. The inset macroscopic image in (a) shows
the aqueous solution of P(NIPAM-co-APUEMA-co-NBD) (1.0 glꢀ1) in
the absence (left) and presence (right) of NO at 208C. Confocal laser
scanning microscopy images (bottom) after incubation of P(NIPAM-
co-APUEMA-co-NBD) with MRC-5 cells for 5 h (A: fluorescent channel,
B: bright-field channel, C: merged bright-field and fluorescent chan-
nel).
Received: March 9, 2014
Published online: June 4, 2014
Keywords: biomimetic polymers · fluorescent probes ·
.
nitric oxide · self-assembly · thermoresponsive polymers
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