Intracellular nitric oxide delivery from stable NO-polymeric nanoparticle carriers

Article abstract of DOI:10.1039/c2cc37181b

The encapsulation of S-nitrosoglutathione into polymeric nanoparticles substantially improves NO stability in aqueous media without affecting the efficacy of intracellular delivery. The combination of nano-NO delivery and chemotherapy has been found to enhance antitumour activity of chemotherapeutics, as demonstrated using preliminary in vitro experiments with neuroblastoma cells.

Full text of DOI:10.1039/c2cc37181b

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Intracellular nitric oxide delivery from stable NO-polymeric
nanoparticle carrierswz
Hien T. T. Duong, Zulkamal M. Kamarudin, Rafael B. Erlich, Yang Li,a
a
a
ab
a
ab
a
a
Mathew W. Jones, Maria Kavallaris, Cyrille Boyer* and Thomas P. Davis*
Received 2nd October 2012, Accepted 25th October 2012
DOI: 10.1039/c2cc37181b
The encapsulation of S-nitrosoglutathione into polymeric nano-
particles substantially improves NO stability in aqueous media
without affecting the efficacy of intracellular delivery. The
combination of nano-NO delivery and chemotherapy has been
found to enhance antitumour activity of chemotherapeutics, as
demonstrated using preliminary in vitro experiments with
neuroblastoma cells.
To improve the administration of NO, a range of small-
molecules capable of decomposition under specific conditions
to release NO have been developed, including nitrate, nitro-
prusside, S-nitrosothiols (RSNOs) and N-diazeniumdiolates
1
0
(NONOates). Among these NO donors, RSNO and NON-
Oates are the most widely used for NO delivery. Unfortu-
nately, these small molecules lack both stability and specificity.
For example, NONOate compounds have a half-life of just a
few minutes at 25 1C. To improve the biodistribution and
stability, NONOate donors have previously been conjugated
to linear polymers or inorganic nanoparticles. In recent work,
NONOate has been successfully conjugated to different poly-
In 1992, nitric oxide (NO) was proclaimed ‘‘Molecule of the
Year’’ by Science Magazine as it has been found to have wide
1
ranging biological effects. NO is an important cellular signalling
molecule and a deficiency of NO has been associated with a number
1
1
2
of diseases, including diabetes, liver fibrosis, cardiovascular
3
meric systems, including a matrix of ethylene/vinylacetate,
8b,12
However, one possible
8
b
star polymers and micelles.
4
5
6
illness, neurodegenerative diseases and several cancers. NO
delivery has been shown to effectively reduce the proliferation
drawback of NONOate compounds is the potential toxicity
of the parent compounds as well as the carcinogenic secondary
amine by-product of NONOate generated during the release of
7
of several cancer cells. In addition, recent studies have shown
the potential of the direct use of NO as a chemotherapy drug
or in combination with chemotherapy agents (cisplatin and
doxorubicin) to enhance their cytotoxicity in a variety of
1
2a,13
NO.
In the meantime, RSNOs are more attractive, as it is
an endogenous class of NO donors.
8
In this communication, we report for the first time a straight-
forward and versatile method to conjugate nitrosoglutathione
different cancer cell lines. Co-delivery of NO and chemotherapeutic
6,8a
agents can also overcome drug resistance in cancer. For example,
Kuppusamy and co-workers demonstrated that NO delivery could
(GSNO) to polymeric nanoparticles. The aqueous stability of
9
overcome cisplatin resistance in ovarian cancer cells. Finally,
GSNO-macromolecular donors and subsequent NO release (in a
reducing environment) were investigated in solution using a
range of techniques (UV-visible, Raman, and fluorescence micro-
scopy). Finally, we demonstrate that the delivery of cisplatin in
combination with NO using nanocarriers using in vitro experi-
ments on a neuroblastoma BE(2)-C cell line has shown to
enhance the antitumor activity of chemotherapeutic agents.
Diblock copolymers were synthesized using reversible addition
another potential application of co-delivery of NO and drugs
6
is to enhance the EPR effect in tumors.
However, the delivery of NO is challenging, as non-systemic
administration is relatively difficult. Currently, NO gas is used
in the clinic for the treatment of respiratory distress syndrome
(
(
RDS) and persistent pulmonary hypertension of the newborn
PPHN). However, delivering NO to organs other than the
1
4
fragmentation chain transfer (RAFT) polymerization as follows:
oligoethylene glycol-methacrylate (OEG-MA) was polymerized in
the presence of (4-cyanopentanoic acid)-4-dithiobenzoate as a
lungs is relatively difficult, as NO is an extremely reactive
molecule that can react with oxygen (and other gases) resulting
in a short half-life time in the body (less than 5 minutes).
0
chain transfer agent and 2,2 -azobisisobutylonitrile (AIBN) as
an initiator. After purification, the polymers were chain extended
with 2-vinyl-4,4-dimethyl-5-oxazolone monomer (VDM) yielding
diblock copolymers (Scheme 1). At the end of the polymerization
(VDM conversion around 60%, as determined by NMR), the
copolymers were purified by precipitation in petroleum ether three
times to remove unreacted VDM. GPC analysis confirmed
successful chain extension (Fig. S1 in the ESIz) with good control
of the polydispersity index and molecular weight (Table S1).
a
Australian Centre for NanoMedicine (ACN), School of Chemical
Engineering, The University of New South Wales, Sydney,
NSW 2052, Australia
Children’s Cancer Institute Australia, Lowy Cancer Research Centre,
b
The University of New South Wales, Sydney, NSW 2052, Australia.
E-mail: t.davis@unsw.edu.au, cboyer@unsw.edu.au
w This article is part of the ChemComm ‘Emerging Investigators 2013’
themed issue.
z Electronic supplementary information (ESI) available. See DOI:
1
1
0.1039/c2cc37181b
H NMR (Fig. S2 and S3, ESIz) and FTIR (Fig. S4 in the ESIz)
This journal is c The Royal Society of Chemistry 2012
Chem. Commun.

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Fig. 1 Release profile of NO (red line) with NO-nanoparticles and
(
(
blue line) NO donor (GSNO) in different media: (A) in water and
B) in water in the presence of ascorbic acid (5 mM). Note: GSNO
Scheme 1 Synthesis of NO-functionalized polymeric nanoparticles
concentration was fixed at 0.4 mM as determined by UV-visible.
for NO delivery.
aqueous solution. GSNO has a half-life of one to two days
at room temperature, limiting its storage. We decided to
investigate whether the encapsulation of NO in polymeric
nanoparticles could result in improved stability in solution.
The signals at 340 nm and 544 nm were monitored by UV
visible spectroscopy. Fig. 1 shows the release of NO from free
GSNO and GSNO encapsulated in polymeric nanoparticles.
The data show that GSNO within nanoparticles has a sig-
nificant enhancement of stability in aqueous solution, with a
half-life time extended to over 14 days (Fig. 1). The rigidity
and hydrophobicity of the nanoparticle core result in a
significant improvement in GSNO stability. GSNO decom-
position yields a disulfide from the intermolecular reaction of
S-nitrosothiols with a thiyl radical generated by homolytic
fission of the sulfur–nitrogen bond (Scheme S1 in the ESIz), a
reaction that is retarded by compartmentalization of S–NO in
polymeric nanoparticles.
spectroscopy confirmed VDM incorporation by the presence
1
ꢀ
of characteristic signals at 1.4 ppm and at 1820 cm , respec-
tively. The proportion of VDM was calculated using the
2
signals at 1.4 ppm and the CH O ester of OEGMA (Table S2).
The azlactone groups were used to attach nitrosoglu-
tathione (GSNO, a common NO donor) to the polymeric
chains. The amino group of GSNO was conjugated to the
azlactone in a basic aqueous solution (in the presence of a
small amount of triethylamine to catalyse the addition of
amine onto azlactone groups) overnight. Azlactone groups
are similar in reactivity to common activated esters (such as
NHS or pentafluorophenyl ester) and are compatible with
1
5
radical polymerizations. The functionalization reaction was
optimized using VDM and GSNO, as monitored by mass
spectroscopy (Fig. S5 in the ESIz). Similar reaction conditions
were then applied to the conjugation of GSNO into polymeric
nanoparticles (formed from the polymer chains assembled in
aqueous media). All the reactions with GSNO were carried out
in the dark to avoid the degradation of GSNO by light. The
successful attachment of GSNO was confirmed using several
techniques: FTIR, NMR and UV-visible spectroscopy. Firstly,
FTIR spectroscopy was invoked to monitor the conversion of the
The release of NO can be stimulated under different specific
conditions, including the application of light, pH or redox.
For intracellular NO release, a convenient mechanism is
reaction with a reducing agent, such as glutathione (GSH),
ascorbic acid or with metallic ions, such as copper and iron. In
this study, ascorbic acid (5 mM) was chosen to mimic the
reductive cellular environment (cytosol). The release of NO
from the nanoparticles has been confirmed using the Griess
Reagent (see ESIz). The release of NO was also monitored by
a decrease in the absorption at 340 nm observed by UV-vis
spectroscopy. As a comparison, GSNO was also tested under
similar conditions. Fig. 1B shows a comparable release profile
for both free GSNO and GSNO-nanoparticles versus time.
Indeed, over 90% of NO was released from the nanoparticles
after 24 h in the presence of ascorbic acid (5 mM). To confirm
that the release mechanism in the nanoparticles was similar to
the expected mechanism for GSNO, Raman spectroscopy was
invoked to confirm the formation of thiols and disulfides (due
to oxidation of thiols by oxygen, see Scheme S1 in ESIz) after
release of NO. After ascorbic acid treatment, a new signal at
500 nm attributed to the disulfide bond was found to be
ꢀ
1
azlactone groups (signal at 1820 cm , Fig. S6 in the ESIz). After
purification (using dialysis against water) and freeze drying, the
ꢀ1
signal at 1820 cm disappears while new signals appear at 1250,
ꢀ1
1
460 and 3300 cm attributable to S–NO and N–H groups,
respectively. NMR analysis reveals a shift of the azlactone signals
at 1.4 ppm to 1.5 ppm consistent with azlactone ring opening.
Finally, UV-visible spectroscopy confirmed the presence of
S–NO bonds at 340 nm and 544 nm after conjugation (Fig. S9
1
6
in the ESIz).
Dynamic light scattering (DLS) and transmission electron
microscopy (TEM) were employed to determine the size of the
nanoparticles before and after conjugation (Fig. S7A). DLS data
indicated a size of around 40 nm (before conjugation, Fig. S8 in
the ESIz) in accord with the sizes determined by TEM. After
conjugation with GSNO, TEM micrographs indicated a slight
increase in size (50 nm). It is interesting to note, during TEM
analysis, that the GSNO containing nanoparticles were degraded
when exposed to the electron beam and the formation of the
bubbles, due to the release of NO, was observed. The release of
NO from the core of the nanoparticles led to the formation of
hollow nanoparticles (Fig. S7B).
1
7
present, in good agreement with the literature (Fig. S10 in
the ESIz).
The cytotoxicity of the nanoparticles was tested on normal
lung fibroblast MRC5 and neuroblastoma cells using Alamar
Blue Assay. The nanoparticles were shown to be non-toxic
ꢀ
1
over the concentration range of 0.01 mg to 10 mg mL
(Fig. S11 in the ESIz). This result is in accord with previous
One of the major drawbacks of GSNO compounds (and all
the primary S-nitrosothiol donors) is their poor stability in
1
8
studies on the toxicity of RAFT polymer based POEG-MA.
Chem. Commun.
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We obtained a similar value for the IC50 of cisplatin for both cell
lines to that obtained without pre-treatment (see Table S3 in the
ESIz).
In this communication, we report a straightforward method
to prepare NO-polymeric nanoparticles, with enhanced NO
stability in aqueous media. The NO-release half-life time was
extended by a factor of 5. The NO-nanoparticles were non-toxic
and could efficiently release NO intracellularly. The application
of NO-nanoparticles, in combination with cisplatin, resulted in
synergistic cytotoxicity to neuroblastoma cancer cells (by a
factor of 5 over cisplatin application alone). In contrast, no
enhanced cytotoxicity was observed when dual NO/cisplatin
administration was tested on a non-cancer cell line.
CB is thankful for ARC APD (DP1092640) and Future
fellowship (FT120100096).
Fig. 2 Confocal microscopy of BE(2)-C: (A) non-treated cells, (B) treated
cells with DAF-FM DA and (C) treated cells with NO-nanoparticles
and DAF-FM DA. Note: Additional pictures are included in the ESIz
(Fig. S12).
Table 1 IC50 values of cisplatin for different cells determined with
and without pre-treatment with NO-nanoparticles
Cells
With NO (mM)
Without NO (mM)
MRC-5
BE(2)-C
10.45 (ꢁ2.32)
1.55 (ꢁ0.81)
Note: all toxicity studies have been repeated in 4 distinct experiments
9.86 (ꢁ2.34)
7.13 (ꢁ1.58)
Notes and references
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Following confirmation that nanoparticles can release NO
in vitro, we decided to demonstrate intracellular NO release
0
0
using 4-amino-5-methylamino-2 ,7 -difluorofluorescein diacetate
DAF-FM DA) (Scheme S2 in the ESIz). When this compound
R. O. Cannon, Clin. Chem., 1998, 44, 1809.
(
19
5 K. J. Smith and H. Lassmann, Lancet Neurol., 2002, 1, 232.
reacts with NO, it emits green fluorescence. BE(2)-C cells were
incubated with the DAF-FM DA probe and then treated with
NO-nanoparticles for 2 h. Green fluorescence was observed from
confocal microcroscopy, confirming the release of NO (Fig. 2C).
Two control experiments were carried out: (i) the cells were treated
only with DAF-FM DA and (ii) the cells were treated with GSH
nanoparticles and DAF-FM DA. A weak fluorescence signal can
be observed for the cells treated only with the probe (Fig. 2B) or
with GSH functionalized nanoparticles and with the probe (Fig.
S13 in ESIz). Green fluorescence is negligible for cells without
treatments (DAF-FM DA and NO-nanoparticles) (Fig. 2A).
As described vide supra, the delivery of NO in combination
with chemotherapy drugs is of considerable interest. We
decided to study the effect of nanoparticle delivery of NO in
combination with cisplatin on neuroblastoma cells (BE(2)-C
cells). Prior to the treatment of cisplatin, the cells were
pre-treated with NO nanoparticles for 8 h. Cell toxicity was
measured using an Alamar Blue assay.
6
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The cells pre-treated with NO-nanoparticles proved to be more
sensitive to cisplatin treatment compared to the non-NO-treated
cells. The IC50 of cisplatin with the pre-treated NO cells is around
1
5 H. T. Ho, M. E. Levere, D. Fournier, V. Montembault, S. Pascual
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5
times lower than the non-pre-treated ones (Table 1). The IC50 of
cisplatin for BE(2)-C cells are slightly lower than reported
2
0
literature values. Such a synergistic effect (NO with cisplatin)
has been reported for other cancer cells using NONOate donors.
It has been observed that the IC50 can be decreased over 60 times
16 J.-W. Park and G. Means, Arch. Pharmacal Res., 1989, 12, 257.
1
7 G. M. Walsh, D. Leane, N. Moran, T. E. Keyes, R. J. Forster,
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1
2
1
when NONOate/cisplatin is combined. As a comparison, we
decided to investigate the effect of NO-nanoparticles and cisplatin
using non-cancerous fibroblast cells (MRC-5 cells). Interestingly,
we did not observe a significant difference between the IC50 of NO
treated and non-treated cells (Table 1) for MRC-5. The IC50 value
obtained without pre-treatment with NO is in good agreement
with the values reported in the literature (9.5 mM obtained by
1
2
9 E. Planchet and W. M. Kaiser, J. Exp. Bot., 2006, 57, 3043.
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2
MTT assay). The synergistic effect of GSH functionalized
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2
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This journal is c The Royal Society of Chemistry 2012
Chem. Commun.