Controlled nitric oxide delivery platform based on S-nitrosothiol conjugated interpolymer complexes for diabetic wound healing

Article abstract of DOI:10.1021/mp900237f

Nitric oxide (NO) is known to play a critical role in enhancing wound healing as topical NO administration has demonstrated enhanced wound healing in diabetic animal models. However, this approach has been limited by the short duration of NO release, short half-life of NO, and instability of available NO donors. To overcome these deficiencies, we have developed a new NO delivery platform based on grafting S-nitrosothiols (RSNOs), derived from endogenous glutathione (GSH) or its oligomeric derivatives, phytochelatins (PCs), onto poly(vinyl methyl etherco-maleic anhydride) (PVMMA), and their subsequent formation of interpolymer complexes with poly(vinyl pyrrolidone) (PVP). Such interpolymer complexes provide controlled release of NO for an extended duration (>10 days) and exhibit enhanced stability in the solid state over that of free RSNOs. The existence of intermolecular hydrogen bonding in such complexes and the formation of disulfide bonds following the NO release have been confirmed by FTIR and Raman. Preliminary wound healing study in a diabetic rat model demonstrates that, with a single topical application, the present controlled release NO delivery system can effectively accelerate wound closure as compared with the control (p < 0.05). The results suggest that the present NO releasing interpolymer complexes could be potentially useful for diabetic wound healing.

Full text of DOI:10.1021/mp900237f

articles
Controlled Nitric Oxide Delivery Platform Based on
S-Nitrosothiol Conjugated Interpolymer Complexes for
Diabetic Wound Healing
Yan Li and Ping I. Lee*
Department of Pharmaceutical Sciences, Leslie Dan Faculty of Pharmacy, UniVersity of
Toronto, Toronto, Ontario M5S 3M2, Canada
Received September 20, 2009; Revised Manuscript Received December 7, 2009; Accepted
December 23, 2009
Abstract: Nitric oxide (NO) is known to play a critical role in enhancing wound healing as topical
NO administration has demonstrated enhanced wound healing in diabetic animal models.
However, this approach has been limited by the short duration of NO release, short half-life of
NO, and instability of available NO donors. To overcome these deficiencies, we have developed
a new NO delivery platform based on grafting S-nitrosothiols (RSNOs), derived from endogenous
glutathione (GSH) or its oligomeric derivatives, phytochelatins (PCs), onto poly(vinyl methyl ether-
co-maleic anhydride) (PVMMA), and their subsequent formation of interpolymer complexes with
poly(vinyl pyrrolidone) (PVP). Such interpolymer complexes provide controlled release of NO
for an extended duration (>10 days) and exhibit enhanced stability in the solid state over that
of free RSNOs. The existence of intermolecular hydrogen bonding in such complexes and the
formation of disulfide bonds following the NO release have been confirmed by FTIR and Raman.
Preliminary wound healing study in a diabetic rat model demonstrates that, with a single topical
application, the present controlled release NO delivery system can effectively accelerate wound
closure as compared with the control (p < 0.05). The results suggest that the present NO
releasing interpolymer complexes could be potentially useful for diabetic wound healing.
Keywords: Nitric oxide; controlled release; interpolymer complex; S-nitrosothiols; diabetic wound
healing
NO delivery in cardiovascular diseases, respiratory diseases,
cancer treatment, antimicrobial therapy, wound healing, and
functional biomedical devices have been proposed and
explored.4-8 Among the numerous benefits of NO, its critical
Introduction
Nitric oxide (NO) is a ubiquitous biological mediator
converted endogenously from L-arginine by nitric oxide
synthases (NOS). The key physiological roles of NO in
cardiovascular, nervous and immune systems have been
identified and elucidated in recent years.1-3 As a result, a
number of potential therapeutic applications of exogenous
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X. D.; He, Y. J.; Huang, S. Y.; Xie, K. P. Direct demonstration
of negative regulation of tumor growth and metastasis by host-
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* To whom correspondence should be addressed. Mailing address:
Leslie Dan Faculty of Pharmacy, University of Toronto, 144
College Street, Toronto, Ontario M5S 3M2, Canada. Tel: +1-
416-946-0606. Fax: +1-416-978-8511. E-mail: ping.lee@
utoronto.ca.
(1) Barbato, J. E.; Tzeng, E. Nitric oxide and arterial disease. J. Vasc.
Surg. 2004, 40, 187–193.
(2) Toda, N.; Okamura, T. The pharmacology of nitric oxide in the
peripheral nervous system of blood vessels. Pharmacol. ReV. 2003,
55, 271–324.
254 MOLECULAR PHARMACEUTICS VOL. 7, NO. 1, 254–266
10.1021/mp900237f  2010 American Chemical Society
Published on Web 12/23/2009

Controlled Nitric Oxide DeliVery Platform
articles
role in the overall wound healing process involving granula-
tion tissue formation, epidermal migration, collagen deposi-
tion and angiogenesis has motivated the pursuit of topical
administration of NO and NO donors in promoting the
healing of diabetic ulcers where impaired wound healing has
been linked to NO and NOS deficiencies at the wound
site.9-11 Such localized and topical delivery of NO is
particularly advantageous as NO is only targeted to the
specific wound site without eliciting a systemic load.
However, these modes of treatment have been limited by
the short duration of NO release, short half-life of NO in
the physiological fluid, and instability of available NO
donors.9,12,13
A range of NO donors derived from two major families
of NO precursors such as diazeniumdiolates (NONOates) and
S-nitrosothiols have been studied extensively.14-16 In an
attempt to prolong the NO release for localized and topical
delivery, NONOates have been incorporated into hydropho-
bic polymers such as polyurethane and poly(vinyl chloride)
to provide extended NO release for a duration of from several
hours up to a week.17-19 However, the clinical utility of
NONOates has been limited by the potential toxicity of the
parent compound and byproduct of NONOate metabolism,
particularly the possibility of forming carcinogenic secondary
nitrosamines.20,21 Although C-based NONOates have been
proposed to avoid the formation of carcinogenic nitrosoam-
ines,22 not all such NONOates can release NO spontane-
ously,23 nor can they be readily adaptable to wound healing
applications.24
On the other hand, S-nitrosothiols (with generic structure,
RSNO) represent an important and a more desirable class
of NO donors because they occur endogenously as S-
nitrosoglutathione (GSNO) or as RSNOs of sulfur-containing
peptides and proteins.15,25 However, the stability of these
RSNOs is often less than desirable as the S-NO bond is
both thermally and photolytically labile, and susceptible to
homolytic cleavage catalyzed by metal ions leading to the
rapid release of NO with the corresponding formation of
disulfides,26 thus limiting their suitability for localized and
topical delivery of NO. In order to improve the stability and
prolong the effective duration of RSNOs, a number of
approaches have been pursued. One strategy involves the
modification of molecular structure of low-molecular weight
RSNOs to increase their lipophilicity,27 or to impart pH-
controlled NO release properties.28 These synthetic RSNOs
and their decomposition products often involve chemical
moieties of unknown pharmacological and toxicological
nature thereby limiting their acceptability for in vivo ap-
plications. Another approach involves conjugating NO to
larger peptides and proteins such as bovine serum albumin
(BSA) or PEG-conjugated BSA through the cysteine
residues.29,30 In addition to rapid NO release, another major
drawback of these NO donors is their low level of achievable
NO loading due to the limited cysteine content of BSA.
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Thornton, F. J.; Barbul, A. Diabetes-impaired healing and reduced
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(23) http://home.ncifcrf.gov/lcc/nitricoxide/NOdonors.asp.
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Cardounel, A. J.; Villamena, F. A.; Wang, P. G. 4-Aryl-1,3,2-
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Alternatively, synthetic polymers physically blended with
RSNO have also been evaluated as NO delivery vehicles.
In this regard, de Oliveira and co-workers incorporated NO
donors such as GSNO and/or S-nitroso-N-acetylcysteine
(SNAC) into films and gels based on water-soluble polymers
such as poly(vinyl alcohol), poly(vinyl pyrrolidone), or
Pluoronic F127 hydrogel, and evaluated such physical blends
for topical NO delivery.31-33 Their animal results suggest
that daily repeated application of GSNO-blended hydrogels
during the early phases of cutaneous wound repair may
accelerate wound closure and re-epithelialization.34 However,
these physically blended NO donor systems still inherit the
same drawbacks of free RSNOs such as rapid NO release
and inactivation upon contact with physiological fluids due
to thermal or metal ion catalyzed degradation. As a result,
the delivery of NO from these existing RSNO based systems
cannot be maintained for any extended duration, generally
not more than several hours, thus requiring frequent reap-
plication from freshly prepared systems.
lowed by S-nitrosation in a gaseous NO/O2 mixture.36 The
resulting hydrophobic polymeric NO donors provided local-
ized NO release and showed enhanced local vasodilation
when applied to the skin. Although such covalent anchoring
of SNO groups has provided some degree of sustained release
of NO, the achievable release duration remained relatively
short (up to only 24 h). In addition, the complexities of their
synthesis and the unknown toxicity of many of the building
blocks and degradation products make it more difficult for
these proposed polymeric NO donors to gain quick clinical
utility. Therefore, there remains a need for a facile prepara-
tion of stable, S-nitrosothiol derived polymeric NO donors
based on biocompatible and pharmaceutically acceptable
components, capable of achieving effective loading and
durable release of NO, suitable for a variety of clinical
applications.
In this study, we report the synthesis of new phytochelatin-
derived NO precursors (S-nitrosophytochelatins or S-nitros-
oPCs) and the subsequent formation for the first time of a
novel series of polymeric NO donors based on interpolymer
complexes containing immobilized RSNOs (GSNO and
S-nitrosoPCs) stabilized in a physically cross-linked polymer
network. More specifically, RSNO precursors covalently
conjugated to a maleic anhydride copolymer, poly(vinyl
methyl ether-co-maleic anhydride) (PVMMA), are further
stabilized in a physically cross-linked polymer network
through intermolecular hydrogen bonding with poly(vinyl
pyrrolidone) (PVP) in the solid state. Here, the existence of
noncovalent interactions such as hydrogen bonding plays an
important role in the self-assembly and formation of inter-
polymer complexes in solution as well as in the solid state.37
Similar to the structure of glutathione (GSH) (Scheme 1A),
phytochelatins (PCs) (Scheme 1B) are a family of oligomeric
derivatives of GSH having the primary structure of (γ-Glu-
Cys)n-Gly, where n ) 2-11, produced by plants to chelate
excess metal ions.38 Recently, these plant derived peptides
have been utilized as biocompatible coatings to modulate
the surface properties of CdSe/ZnS nanocrystals.39 The
interpolymer complexes obtained in the present study are
capable of providing continuous and prolonged NO genera-
tion with enhanced RSNO storage stability. Furthermore, all
components of the present system are either endogenous in
origin (e.g., GSH and GSNO) or biocompatible (e.g., PCs),
or have prior history of use in humans as well as in approved
drug products (e.g., PVMMA and PVP). In addition to
In order to achieve a meaningful extension of NO release
duration, the macromolecular prodrug approach with S-
nitrosothiol groups covalently attached to the polymer
backbone appears to be a viable strategy for enhancing the
stability of RSNOs, as well as in controlling the release
duration of NO. In this regard, Bohl and West synthesized
S-nitrosocysteine (CysNO)-derived PEG-hydrogels which
exhibited reduced platelet adhesion and smooth muscle cell
proliferation in cell culture.35 Despite this seemingly en-
couraging result, most of the NO release occurred in the first
4 h and the achievable level of NO loading in these hydrogels
remained very low (<1% w/w, based on stoichiometric data).
On the other hand, de Oliveira and co-workers synthesized
hydrophobic sulfhydryl (-SH)-containing polyesters fol-
(30) Katsumi, H.; Nishikawa, M.; Yamashita, F.; Hashida, M. Devel-
opment of polyethylene glycol-conjugated poly-S-nitrosated serum
albumin, a novel S-nitrosothiol for prolonged delivery of nitric
oxide in the blood circulation in ViVo. J. Pharmacol. Exp. Ther.
2005, 314, 1117–1124.
(31) Seabra, A. B.; Da Rocha, L. L.; Eberlin, M. N.; de Oliveira, M. G.
Solid films of blended poly(vinyl alcohol)/poly(vinyl pyrrolidone)
for topical S-nitrosoglutathione and nitric oxide release. J. Pharm.
Sci. 2005, 94, 994–1003.
(32) Shishido, S. M.; Seabra, A. B.; Loh, W.; de Oliveira, M. G.
Thermal and photochemical nitric oxide release from S-nitro-
sothiols incorporated in Pluronic F127 gel: potential uses for local
and controlled nitric oxide release. Biomaterials 2003, 24, 3543–
3553.
(33) Seabra, A. B.; Fitzpatrick, A.; Paul, J.; de Oliveira, M. G.; Weller,
R. Topically applied S-nitrosothiol-containing hydrogels as ex-
perimental and pharmacological nitric oxide donors in human skin.
Br. J. Dermatol. 2004, 151, 977–983.
(36) Seabra, A. B.; da Silva, R.; de Oliveira, M. G. Polynitrosated
polyesters: preparation, characterization, and potential use for
topical nitric oxide release. Biomacromolecules 2005, 6, 2512–
2520.
(37) Jiang, M.; Li, M.; Xiang, M.; Zhou, H. Interpolymer complexation
and miscibility enhancement by hydrogen bonding. AdV. Polym.
Sci. 1999, 146, 121–196.
(38) Rauser, W. E. Phytochelatins and related peptides: structure,
biosynthesis, and function. Plant Physiol. 1995, 109, 1141–1149.
(39) Pinaud, F.; King, D.; Moore, H. P.; Weiss, S. Bioactivation and
cell targeting of semiconductor CdSe/ZnS nanocrystals with
phytochelatin-related peptides. J. Am. Chem. Soc. 2004, 126,
6115–6123.
(34) Amadeu, T. P.; Seabra, A. B.; de Oliveira, M. G.; Costa, A. M. A.
S-Nitrosoglutathione-containing hydrogel accelerates rat cutaneous
wound repair. J. Eur. Acad. Dermatol. Venereol. 2007, 21, 629–
637.
(35) Bohl, K. S.; West, J. L. N itric oxide-generating polymers reduce
platelet adhesion and smooth muscle cell proliferation. Biomate-
rials 2000, 21, 2273–2278.
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Scheme 1. Chemical Structure of (A) Glutathione (GSH)
and (B) Phytochelatin
molar ratio of PC2 and PC5 to NaNO2 was 1:2 and 1:5 to
account for the increasing number of thiol groups on PC2
and PC5, respectively.
Conjugation of RSNOs to PVMMA. The facile attach-
ment of RSNOs to PVMMA was achieved via a heteroge-
neous reaction of RSNO with PVMMA. In the case of
GSNO, 500 mg of PVMMA was first dissolved homoge-
neously in 10 mL of acetone. The above GSNO stock
solution (2 mL) was then added dropwise into the PVMMA
solution under stirring in an ice bath. Subsequently, the
mixture was poured onto a Teflon dish and acetone was
removed by either vacuum drying or air-drying in a fume
hood at room temperature and protected from light. The
obtained GSNO-PVMMA in the form of pink powder with
11.86 wt % GSNO loading was collected and stored in a
desiccator. Additionally, a portion of the resultant solution
was kept without drying and protected from light until the
next complexation step. The conjugation of S-nitrosoPC2 and
S-nitrosoPC5 to PVMMA was achieved in an identical
fashion.
Preparation of RSNO-PVMMA/PVP Interpolymer
Complexes. For RSNO-PVMMA/PVP complexes, a 6.36
wt % PVP solution was first prepared in a mixture of 10:1
(v/v) acetone and ethanol. In the case of GSNO-PVMMA,
4 mL of ethanol was added to the GSNO-PVMMA solution
(in 10/2 acetone/0.1 N HCl) prior to the complex formation.
Subsequently, a measured amount of the PVP solution was
quickly poured into the GSNO-PVMMA solution under
vigorous stirring in an ice bath. As the complex formation
took place through intermolecular hydrogen bonding, the
viscosity of the resultant mixture increased significantly
giving rise to a pink gel-like product with the extent of
gelation varying with composition. Although the PVMMA/
PVP weight ratio can be varied by adjusting the volume of
PVP solution, only 1:1 ratio was selected as an example for
this study. The polymer complex so obtained, with 6.3 wt
% GSNO loading, was air-dried, mixed with dry ice and
pulverized in a laboratory grinding mill. The resulting pink
powder was stored in amber glass vials prior to subsequent
in Vitro characterization and animal studies. Likewise,
S-nitrosoPC2-PVMMA/PVP (1:1) and S-nitrosoPC5-
PVMMA/PVP (1:1) were also prepared following identical
procedures.
UV-Vis Spectra. The presence of the S-NO group in
both RSNO and RSNO-conjugated PVMMA/PVP complex
was verified via the appearance of characteristic absorbance
maxima at 336 and 545 nm in the UV and visible ranges
corresponding to π f π*and nN f π*electronic transition
of the S-NO bond, respectively.32,36 UV-vis spectra were
recorded in the wavelength range of 200-800 nm at room
temperature on a Cary 50 UV-vis spectrophotometer (Varian
Inc., Palo Alto, CA).
FTIR Spectra. The attachment of GSNO to PVMMA and
hydrogen bonding interaction between GSNO-PVMMA and
PVP were characterized by Fourier transform infrared (FTIR)
on a universal attenuated total reflectance (ATR) Spectrum-
one Perkin-Elmer spectrophotometer (Perkin-Elmer, Walth-
presenting the preparation and characterization of these novel
RSNO-PVMMA/PVP interpolymer complexes, we also
report the use of a diabetic rat model to assess the benefit of
one such polymeric NO delivery system in wound healing.
Materials and Methods
Materials. Reduced glutathione (GSH), sodium nitrite
(NaNO2), sulfanilamide (SULF) and N-(1-naphthyl)ethyl-
enediamine dihydrochloride (NEDD) were obtained from
Sigma-Aldrich Canada (Oakville, ON). Phytochelatins, (γ-
Glu-Cys)n-Gly, with n ) 2 and 5 (PC2 and PC5, respectively)
were purchased from AnaSpec Inc. (San Jose, CA). Poly-
(vinyl methyl ether-co-maleic anhydride) (PVMMA, Gantrez
AN-169, MW: 2,000,000) and poly(vinyl pyrrolidone) (PVP
Plasdone K-90, MW: 1,300,000) were generously provided
by ISP (Wayne, NJ). Other chemicals and solvents of
analytical reagent grade were obtained from Sigma-Aldrich
Canada, and they were used as received unless stated
otherwise. Milli-Q grade (Millipore; Billerica, MA) deionized
water was used for all solutions and buffers.
Synthesis of RSNOs. GSNO was readily prepared by
reacting GSH with equimolar sodium nitrite in an acidic
medium protected from exposure to light. Briefly, to a stirred
ice-cold solution of GSH (154 mg, 0.5 mmol) in 5 mL of
0.1 N HCl was added a portion of NaNO2 (35 mg, 0.5 mmol).
This reaction gave GSNO in a high yield of more than 80%
(equivalent to the fractional conversion of NaNO2 determined
by the Griess Assay as described in the section below on in
Vitro NO release). The resulting GSNO stock solution (red
color) was protected from light with aluminum foil and used
directly without further purification.
The S-nitrosation of phytochelatins (PC2 and PC5) was
carried out in a similar fashion as GSNO, except that the
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Li and Lee
am, MA). The spectra were recorded from 650 to 4000 cm-1.
All spectra were collected at a resolution of 2 cm-1 and were
repeated three times. A background spectrum without any
sample was subtracted from all spectra.
determination of nitrite, a stable decomposition product of
NO, in solution using the Griess assay to provide a
quantitative measure of NO release from various NO donors
has been widely accepted.35,36,42,43
Raman Spectra. Raman spectra were acquired using a
Raman spectroscopy system (LabRam HR800, Jobin Yvon,
France), which is equipped with a liquid-nitrogen-cooled
CCD and a confocal microscope system (Olympus, BX41)
with 3 objectives and a 60× (NA 0.9) water objective. The
325 nm line of a He-Cd UV-laser (Kimmon IK3301R-G)
with a spot size of 1 µm was used as the excitation source.
Samples of GSNO-PVMMA/PVP (1:1) lyophilized powder,
before and after 24 h NO release, were evenly spread on
individual microscope slides. Raman spectra were recorded
at room temperature in the spectral region from 200 to 1200
cm-1 covering the S-S and C-S vibrational modes.
Decomposition Kinetics in Release Medium. The de-
composition kinetics of GSNO-PVMMA/PVP complex
versus that of GSNO in the release medium was studied
employing the same experimental setup, temperature and
light conditions as described in the previous section for the
in Vitro NO release. At time zero, either a GSNO-PVMMA/
PVP complex powder sample or a small aliquot of the GSNO
stock solution having identical GSNO content was mixed
with the release medium and protected from light. After 2
days in the dark, the sample vials were switched to
continuous light exposure for the remainder of the experi-
ment. Throughout the entire duration of the experiment, the
release medium was sampled at predetermined time intervals
for a rapid determination of GSNO concentration by the UV
method. These samples of the release medium were protected
from light before and after the UV reading, and immediately
returned to the sample vials for the continuation of the
studies. The change in GSNO concentration in the release
medium due to cleavage of S-N bond from exposure to
different lighting and temperatures conditions was monitored
by following the decay of UV absorbance at 336 nm as a
function of time. This method is specific to changes in the
GSNO concentration and not affected by the degradation
products of NO as the UV absorbance of nitrite and nitrate
is negligibly small between 300 and 400 nm in the µmol
concentration range studied here.
Stability Studies. In one experiment, GSNO-PVMMA/
PVP (1:1) complex powder was stored in glass vials at room
temperature (relative humidity: ∼32%.) without protection
from light for a duration of 6 months. In another experiment,
GSNO-PVMMA/PVP (1:1) complex powder was exposed
to direct UV irradiation from a UV lamp in a Labguard Class
II Biological Safety Cabinet (66 cm height, 15 W UV light)
for 24 h. The NO release profiles from these stability samples
were determined and compared with that of the original
samples.
Additionally, the stability of GSNO, S-nitrosoPC5 and
S-nitrosoPC5-PVMMA/PVP (1:1) complex in PBS in the
presence of copper ion was investigated by monitoring the
decay of UV absorbance at 336 nm in sample solutions
containing a fixed concentration of Cu2+ (36 µM).
In Vitro NO Release. The in Vitro release study was
typically carried out in a scintillation vial by immersing 20
mg of powder sample in 10 mL of 0.1 M phosphate buffered
saline (PBS; pH 7.4) containing 0.1 wt % Na2EDTA for the
duration of the release experiment. The addition of chelating
agent Na2EDTA was to minimize any degradation of RSNO
in the release medium catalyzed by metal ions. Multiple
sample vials were placed on a vial rotator at a rotating speed
of 15 rpm maintained at either room temperature (22 °C) or
37 °C in an incubator under a selected lighting condition.
For continuous light exposure, the samples were exposed to
fluorescent lamps (two USHIO F25T8/741 fluorescent tubes;
4100K) at an intensity of approximately 500 lx at the vial
surface (measured with a light meter). These fluorescent
lamps exhibit a spectral power distribution between 400 and
750 nm with about 55% of the total power output in the
range of 530-620 nm.40 The visible spectral range of
550-600 nm has been reported to be responsible for a major
portion of the photoinitiated NO release from S-nitrosothi-
ols.41 For ambient light conditions, the samples were exposed
to a normal day/night cycle in the laboratory with no special
lighting control. To obtain cumulative NO release, the
measurement was performed by consuming individual samples
at predetermined time intervals. The release medium of each
vial was then sampled and the NO release from the polymer
immediately quantified by the standard Griess assay. Briefly,
1 mL of Griess reagent (NEDD) (0.1% w/v) plus 1 mL of
sulfanilamide (1% w/v in 5% w/v H3PO4) at room temper-
ature was incubated with an equal volume (1 mL) of the
release medium. UV-vis absorbance of the resulting solution
was determined at 540 nm, and the total nitrite concentration
In ViWo Wound Healing. The animal study was performed
under an animal protocol approved by the University of
Toronto Animal Care Committee. Briefly, 15 male Sprague-
-
[NO2 ] in the sample solution was calculated from a standard
curve and converted to cumulative NO release expressed as
nmol/mg of sample. Each datum point in the resulting NO
release profile represents the mean of triplicate samples (n
) 3) with the error bar denoting the standard deviation. The
(42) Mowery, K. A.; Schoenfisch, M. H.; Saavedra, J. E.; Keefer, L. K.;
Meyerhoff, M. E. Preparation and characterization of hydrophobic
polymeric films that are thromboresistant via nitric oxide release.
Biomaterials 2000, 21, 9–21.
(40) http://www.ushio.com/files/specs/Ultra8T8UBend.pdf.
(41) Frost, M. C.; Meyerhoff, M. E. Controlled photoinitiated release
of nitric oxide from polymer films containing S-nitroso-N-acetyl-
DL-penicillamine derivatized fumed silica filler. J. Am. Chem. Soc.
2004, 126, 1348–1349.
(43) Nims, R.; Cook, J.; Krishna, M.; Christodoulou, D.; Poore, C.;
Miles, A.; Grisham, M.; Wink, D. Colorimetric assays for nitric
oxide and nitrogen oxide species formed from nitric oxide stock
solutions and donor compounds. Methods Enzymol. 1996, 268,
93–105.
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Scheme 2. (A) Conjugation of RSNO to PVMMA and (B)
Hydrogen Bonding Interaction between RSNO-PVMMA
and PVP
Dawley rats (Charles River, Montreal, QC) were acclimatized
for one week, given food and water ad libitum. Seven days
before wounding, the animals were injected intraperitoneally
(IP) with streptozotocin (60 mg per kg body weight in citrate
buffer 0.1 mol/L, pH 4.5) to induce diabetes. Evidence of
diabetes was confirmed by blood glucose levels greater than
14 mmol/L and frequent urination. Animals not achieving the
diabetic state after 24 h were reinjected with streptozotocin.
Subsequently, animals with blood glucose level still remaining
below14 mmol/L were excluded from the study. After the
induction of diabetics, the blood glucose level was monitored
twice a week to ensure that the diabetic state was maintained
throughout the entire wound healing experiment.
For the surgery, animals were weighed and assigned to
two groups (5 for the control and 7 for the test group; three
excluded). The following procedures were conducted while
animals were anesthetized with isoflurane inhalation. First,
the dorsal surface hair was removed by shaving and the skin
washed with povidone-iodine solution and 70% alcohol.
Subsequently, a full thickness excisional wound was created
by removal of the skin and panniculus carnosus using an 8
mm biopsy punch. Rats were given analgesic (ketoprofen, 5
mg/kg, sc) immediately after surgery. At the wound sites,
the control group was treated with 20 mg of blank PVMMA/
PVP complex powder without GSNO loading, while the test
group was treated with 20 mg of GSNO-PVMMA/PVP (1:
1) complex powder (6.3 wt % GSNO loading). The GSNO
loading level and the amount of complex powder were
selected to ensure the delivery of µmol quantity of GSNO
per application and the complete coverage of each wound
area by the powder. All polymer powders quickly adhered
to the wound tissue with the addition of two drops of sterile
saline. After a single application of the polymer powder,
wounds were covered with transparent Tagederm dressings
(3M; St. Paul, MN) sealed at the edges with tincture of
benzoin (Benzoin Simple Tincture NF; Xenex Laboratories,
Ferndale, WA). Afterward, animals were transferred to
individual cages and maintained on a standard diet, allowed
access to water ad libitum. All animal experiments were
conducted under ambient light conditions.
Results and Discussion
Synthesis and Characterization. GSNO, S-nitrosoPC2
and S-nitrosoPC5 were prepared via nitrosation of thiols
according to the following reaction scheme:
+
H3O
RSH + HNO2
8 RSNO + H2O
(1)
This reaction is generally very rapid and quantitative.
Although GSNO is a known NO precursor, phytochelatin-
derived NO precursors synthesized here such as S-nitrosoPC2
and S-nitrosoPC5 are new. One key advantage of the present
NO precursors over other systems is that GSH and phytoch-
elatins are short, cysteine rich peptides, which can provide
high levels of NO loading after nitrosation.16,17
Following the reaction scheme of Scheme 2A, S-nitros-
oPC2 and S-nitrosoPC5 have been conjugated to PVMMA
also for the first time to form a novel series of polymeric
NO precursors. A notable characteristic of maleic anhydride
copolymers such as PVMMA is the high reactivity between
the anhydride moieties and primary amine groups. Covalent
modification of proteins with maleic anhydride was reported
as early as the 1960s;44 such a reaction can be beneficially
performed via surface chemistry following the interfacial
presentation of bioactive molecules.45,46 In our case, the
conjugation of RSNO to PVMMA was achieved via a
heterogeneous reaction because RSNO has to be dissolved
At selected time points during the postwounding period,
the animals were anesthetized by isoflurane inhalation and
photographs of the wound sites were recorded using a digital
camera; a calibration scale was also recorded with each
photograph. During the first 7 days after wounding, the
Tegaderm dressing was changed daily. Beyond 7 days, the
wounds were no longer covered with dressings. The surface
area of each lesion was quantified from the photographs using
Image-Pro Plus 5.0 software to determine the area of the
open wound. The results are expressed in percentage of initial
wound area as a function of time. For each datum point,
mean and standard deviation were calculated. Statistical
analysis between experimental groups was performed using
unpaired two-tailed Student’s t tests. The confidence limit
was predetermined at an alpha level of 0.05.
(44) Butler, P. J. G.; Harris, J. I.; Hartley, B. S.; Leberman, R. Use of
maleic anhydride for reversible blocking of amino groups in
polypeptide chains. Biochem. J. 1969, 112, 679–689.
(45) Ladavie`re, C.; Lorenzo, C.; Ela¨ıssari, A.; Mandrand, B.; Delair,
T. Electrostatically driven immobilization of peptides onto (maleic
anhydride-alt-methyl vinyl ether) copolymers in aqueous media.
Bioconjugate Chem. 2000, 11, 146–152.
(46) Allard, L.; Cheynet, V.; Oriol, G.; Mandrand, B.; Delair, T.;
Mallet, F. Versatile method for production and controlled polymer-
immobilization of biologically active recombinant proteins. Bio-
technol. Bioeng. 2002, 80, 341–348.
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in 0.1 N HCl whereas PVMMA is only soluble in acetone,
and because acetone and aqueous HCl are precipitating
agents for RSNO and PVMMA, respectively. Therefore, the
grafting reaction takes place only at the interface of RSNO
and PVMMA in solution. Depending on the volume ratio
and concentration of the two components, the RSNO loading
level can be varied. Comparing with other NO delivery
systems,16,17 the abundance of functional anyhydride groups
on the backbone of PVMMA enables the attachment of
RSNOs with grafting degree as high as 50% for GSNO.
The complexation of RSNO-PVMMA and PVP is based
on interpolymeric hydrogen bonding interactions depicted
in Scheme 2B. The formation of interpolymer complexes
via hydrogen bonding between a proton-donating polymer
such as poly(carboxylic acid) and a proton-accepting non-
ionic polymer such as PVP has been extensively investigated
for pharmaceutical purposes.47,48 This is typically ac-
complished by first dissolving each polymer component
separately in a common solvent followed by mixing the
component solutions. The resulting formation of interpolymer
complexes is often manifested by an increase in solution
viscosity or the precipitation of a separate gel phase. In our
study, poly(vinyl pyrrolidone) (PVP), which contains a large
number of pendant pyrrolidone carbonyl groups, is used as
a proton-accepting polymer to complex with the hydrolyzed
portion of PVMMA in RSNO-PVMMA, which acts as a
proton-donating polymer. Since RSNO-PVMMA is formed
in an acetone based solvent and PVP does not dissolve in
pure acetone, a small amount of ethanol was introduced in
the PVP solution to facilitate the complex formation. To
ensure that phase separation does not result from solvent
induced PVP precipitation, a comparable amount of ethanol
was also incorporated into the RSNO-PVMMA solution
before blending with the PVP solution.
Figure 1. (A) UV and (B) visible spectra of (a) pure
PVMMA in acetone, (b) GSNO in aqueous medium, and
(c) GSNO-PVMMA in aqueous medium; and (C)
UV-vis spectra of (a) pure PC5, (b) S-nitrosoPC5, and
(c) S-nitrosoPC5-PVMMA/PVP (1/1) complex in aque -
ous medium.
The presence of S-NO group in both GSNO and GSNO-
conjugated PVMMA is demonstrated via the appearances
of the characteristic absorbance of S-NO bond at λ ) 336
nm and λ ) 545 nm (see Figure 1A,B), corresponding to
the maximum absorption in the UV and visible ranges,
respectively. These have been assigned to the π f π* and
nN f π* electronic transitions of the S-NO bond, respec-
tively. Likewise, in Figure 1C, the presence of an absorbance
peak at λ ) 336 nm verifies the successful synthesis of
S-nitrosoPC5. A similar absorbance spectrum was also
observed with S-nitrosoPC2 (data not shown).
The IR spectra of GSNO, PVMMA, GSNO-PVMMA,
and GSNO-PVMMA/PVP (1:1) complex are shown to-
gether in Figure 2. The small band at 1706 cm-1, originated
from acid dimers likely formed from a trace amount of
carboxylic acid groups in the PVMMA raw material (curve
Figure 2. The FTIR spectra of (a) GSNO, (b) PVP, (c)
PVMMA, (d) GSNO- PVMMA, and (e) GSNO-
PVMMA/PVP (1/1) complex.
(47) Khutoryanskiy, V. V. Hydrogen-bonded interpolymer complexes
as materials for pharmaceutical applications. Int. J. Pharm. 2007,
334, 15–26.
(48) Haddadine-rahmoun, N.; Amrani, F.; Arrighi, V.; Cowie, J. M. G.
Interpolymer complexation in hydrolysed poly(styrene-co-maleic
anhydride)/poly(styrene-co-4-vinylpyridine). Eur. Polym. J. 2008,
44, 821–831.
c), has been replaced by the characteristic ester carbonyl band
at 1724 cm-1 (curves d and e), which can be attributed to
260 MOLECULAR PHARMACEUTICS VOL. 7, NO. 1

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Figure 4. In vitro release profiles comparing NO release
in 0.1 M phosphate buffer saline (containing 0.1 wt %
Na2EDTA) from S-nitrosoPC5-PVMMA/PVP (1/1)
interpolymer complex powder containing 3.24 wt % of
S-nitrosoPC5 at 22 and 37 °C under ambient light
conditions. Each datum point is the mean of three
samples, and the error bar represents the standard
deviation.
Figure 3. In vitro release profiles comparing NO release
in 0.1 M phosphate buffer saline (containing 0.1 wt %
Na2EDTA) from GSNO-PVMMA/PVP (1/1) interpolymer
complex powder containing 6.3 wt % of GSNO at 22
and 37 °C under different lighting conditions. Each
datum point is the mean of three samples, and the error
bar represents the standard deviation.
their esterification with ethanol during the GSNO coupling
process. Another major absorption band at 1642 cm-1
appears in the spectrum of GSNO-conjugated PVMMA
(curve d) which is characteristic of the carbonyl group of an
amide. This provides direct evidence for the acylation
reaction between the anhydride group of PVMMA and the
primary amino group of GSNO. The hydrogen bonding
interaction in the present GSNO-PVMMA/PVP complex
is also reflected in the IR spectra of Figure 2, where a shift
of the carbonyl stretching band in the pyrrolidone ring from
1650 cm-1 in pure PVP (curve b) to 1656 cm-1 in the
GSNO-PVMMA/PVP complex (curve e) is quite evident.
Such an upward shift in carbonyl stretching frequency as a
result of intermolecular hydrogen bonding is well-known in
other miscible polymer blends.49,50
to 10 days or more under ambient light conditions. In addition
to the faster rate and shorter duration of NO release at
elevated temperature (37 °C vs 22 °C), continuous light
exposure also results in a faster NO release as compared
with the ambient light condition where the exposure to light
is of intermittent nature due to the natural day/night cycle.
Nevertheless, as shown in Figure 3, even with faster NO
release under continuous light exposure, the characteristic
feature of sustained NO delivery from the present polymer
complex is still retained with duration of release up to 7 days
or more. The release profiles of Figure 3 represent about
40-50% of total NO release. Additional NO release is still
possible with higher intensity of light irradiation. Further
kinetic experiments relating to this aspect are currently
ongoing.
Effect of Temperature and Light on in Vitro NO
Release. Typical profiles of NO release from the GSNO-
PVMMA/PVP (1/1) complex (with 6.3 wt % GSNO loading)
at 22 and 37 °C under continuous light exposure are shown
in Figure 3, where the total amount of NO release is
normalized to sample weight. For comparison, a correspond-
ing NO release profile at 22 °C under ambient light
conditions is also included in Figure 3. It is clear that the
duration of NO release from the present interpolymer
complex containing immobilized GSNO can be extended up
Similarly, Figure 4 demonstrates the sustained NO release
behavior from the S-nitrosoPC5-PVMMA/PVP (1:1) com-
plex with 3.24 wt % S-nitrosoPC5 loading as a function of
temperature under ambient light condition, where the release
period is similarly extended up to at least 10 days and the
release profiles represent about 60-70% of total NO release.
Moreover, the use of PC5 as the NO donor enables a higher
total amount of NO delivered per mg of sample due to the
presence of more thiol groups on PC5. It is worth noting
that S-nitrosoPC5-PVMMA/PVP (1:1) complexes exhibit
solubility behavior that is quite different from that of
GSNO-PVMMA/PVP (1:1). In our observations, S-nitroso-
PC5-PVMMA/PVP (1:1) appeared to dissolve in phosphate
buffer at room temperature, resulting in a gel-like solution
within hours, whereas the dissociation of the GSNO-
PVMMA/PVP (1:1) complex took several days. This aspect
(49) Lee, J. Y.; Painter, P. C.; Colman, M. M. Hydrogen bonding in
polymer blends. 3. Blends involving polymers containing meth-
acrylic acid and ether groups. Macromolecules 1988, 21, 346–
354.
(50) Xu, X.; Lee, P. I. Programmable drug delivery from an erodible
association polymer system. Pharm. Res. 1993, 10, 1144–1152.
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concentration, curve d of Figure 5 shows some noticeable
thermal decomposition at 37 °C in the dark after 2 days.
On the other hand, curves a and b of Figure 5 show a
gradual buildup of GSNO concentration over the same
time period due to the erosion and slow dissolution of
the GSNO-PVMMA/PVP (1:1) complex sample. In this
case, the dissolution of the complex is complete at around
2 days at both 22 and 37 °C and there is a negligible loss
of GSNO from the GSNO-PVMMA/PVP (1:1) complex
in the release medium when protected from light for a
period of 2 days as confirmed by independent Griess
assays (data not shown). This suggests that the decom-
position kinetics in the release medium in the dark appears
to be less affected by temperature for the present
GSNO-PVMMA/PVP (1:1) complex as compared with
free GSNO.
The subsequent decomposition kinetics mediated by
continuous light exposure and thermal effect is also shown
in Figure 5. It is evident that the light-induced decomposition
of GSNO can be significantly slowed by immobilizing GSNO
in the present interpolymer complex (curves a and b) as
compared with the rapid decomposition of free GSNO
(curves c and d). For example, based on the absorbance data
of Figure 5, around 40% of the conjugated GSNO groups
still remain in the interpolymer complex after 6 days of
continuous light exposure in the solution phase, whereas the
free GSNO is almost depleted after only 4 days. On the other
hand, the decomposition kinetics in the release medium under
continuous light exposure seems to be similarly affected by
temperature for both the GSNO-PVMMA/PVP (1:1) com-
plex and free GSNO.
The decreased decomposition rate of GSNO-PVMMA/
PVP (1:1) complex can be attributed to several factors. It is
known that the NO release from GSNO and related com-
pounds generally involves two steps,51-53 one of which is
due to homolytic bond cleavage via photoexcitation (eq 3);
another involves subsequent reactions of the produced thiyl
radical either directly with GSNO (eq 4) or with oxygen to
produce the peroxy radical, which then reacts with GSNO
to give GSSG (eqs 5 and 6).
Figure 5. Decomposition kinetics of GSNO-PVMMA/
PVP (1/1) interpolymer complex (a and b, 6.3 wt % of
GSNO) versus that of free GSNO (c and d) in 0.1 M
phosphate buffer saline (containing 0.1 wt % Na2EDTA)
at (a, c) 22 °C and (b, d) 37 °C; in the dark for the initial
2 days followed by continuous light exposure for 7
days. Each datum point is the mean of three samples,
and the error bar represents the standard deviation.
is currently being further investigated to delineate the
significance of such a difference in polymer erosion behavior.
Decomposition Kinetics in Release Medium. It is well-
known that homolysis of RSNO forming disulfide bridges
is the main mechanism responsible for its degradation
which can be catalyzed by metal ions (e.g., Cu2+), heat,
and light:51
heat, light, and metal
2RSNO
RSSR + 2NO
(2)
Figure 5 illustrates the decomposition kinetics of GSNO-
PVMMA/PVP (1:1) complex versus that of GSNO in the
release medium at 22 and 37 °C under different lighting
conditions, where the catalytic degradation of GSNO by
metal ions has been minimized by the addition of
Na2EDTA, a chelating agent, in the release medium. A
UV method rather than the Griess assay is employed here
to allow for tracking the real time changes in GSNO
concentration in the release medium from a single sample,
whereas the Griess assay is more time-consuming and
requires the consumption of one sample for each measure-
ment. The UV absorbance reading correlates directly to
the concentration of GSNO in the sample at any given
time. It can be seen from curve c of Figure 5 that GSNO
in PBS at pH 7.4 is quite stable at 22 °C when protected
from light as there is no detectable decomposition during
the initial 2 days. However, with the same initial GSNO
GSNO f GS• + NO•
GSNO + GS• f GSSG + NO•
GS• + O2 f GSOO•
(3)
(4)
(5)
GSOO• + GSNO f GSSG + NO• + O2
(6)
Similarly, upon NO generation in the case of the present
GSNO-PVMMA/PVP (1:1) complex, macromolecular radi-
(52) Sextona, D. L.; Ruganandadmo, R.; Mckenney, N. J.; Mutus, B.
Visible light photochemical release of nitric oxide from S-
nitrosoglutathione: Potential photochemotherapeutic applications.
Photochem. Photobiol. 1994, 59, 463–461.
(53) Wood, P. D.; Mutus, B.; Redmond, R. W. The Mechanism of
Photochemical Release of Nitric Oxide from S-Nitrosoglutathione.
Photochem. Photobiol. 1996, 64, 518–524.
(51) Williams, D. L. H. The chemistry of S-nitrosothiols. Acc. Chem.
Res. 1999, 32, 869–876.
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Figure 6. Molecular scheme depicting the NO release from the RSNO-PVMMA/PVP interpolymer complex network
yielding disulfide bonds in the solid state.
further reinforces the network structure of the complex. Such
disulfide cross-linking would reduce the rate of erosion of
the polymer complex in an aqueous medium, thereby
preventing the polymeric complex from quickly dissolving
in an aqueous medium and consequently further slowing the
diffusion-limited process for the liberation of NO as reflected
in the gradual leveling off of curves a and b of Figure 5
above, thus capable of prolonging the NO release at the local
site of application. Depending on the polymer structure and
state of chain packing, a different controlled release rate can
be obtained by adjusting the RSNO loading, the component
polymer molecular weight and the concentration ratio in the
complex.
This proposed formation of disulfide bonds during NO
release is verified by comparing the Raman spectra of the
GSNO-PVMMA/PVP complex before and after NO release
in Figure 7. The appearance of distinct Raman lines peaking
at 455 cm-1 (S-S stretching) and 630 cm-1 (C-S stretching)
is indicative of the formation of -CSSC-, confirming that
some S-NO groups have been converted to disulfide bonds,
thereby liberating NO. Although -SS- has also been
associatedwithRamanbandsexcitedat514.5nmradiation,55,56
the selection of UV excitation at 325 nm in our Raman study
is more convenient because it avoids the typical fluorescence
background found in samples excited in the visible range.
Stability in the Presence of Copper Ion. Since copper
can directly and catalytically trigger the decomposition of
RSNO in solution,57 the stability of GSNO, S-nitrosoPC5,
and S-nitrosoPC5 conjugated PVMMA/PVP complex in the
cals PVMMA-GS• or PVMMA-GSOO• will form and
recombine with GSNO-PVMMA, leading to the further
liberation of NO and the formation of a dimer linked by the
disulfide bridge. The increased bulkiness through chemical
conjugation of GSNO with the macromolecule PVMMA
leads to a slower diffusion rate (or reduced mobility) of the
PVMMA-GS• or PVMMA-GSOO• radical as well as that
of GSNO-PVMMA compared with the corresponding
species derived from free GSNO. This results in a slower
rate of recombination of these species and therefore a slower
rate of NO release (or GSNO decomposition) from the
current GSNO-PVMMA/PVP (1:1) complex as seen in
Figure 5 (curves a and b). Therefore, compared with other
reported RSNO-linked polymers,36,54 the very high MW of
PVMMA employed in the present study (MW 2,000,000)
further reduces the rate of NO liberation and enhances the
extent of the extended NO release observed here.
NO Release Mechanism. To gain additional insight into
the mechanism of NO release from RSNO-PVMMA/PVP
complexes, a proposed molecular scheme is depicted in
Figure 6. It is envisioned that as the RSNO-PVMMA/PVP
complex is in contact with an aqueous medium, the hydro-
philic components of the polymer complex (hydrolyzed
PVMMA and PVP) would swell, giving rise to increased
polymer chain mobility, thus providing more opportunity for
the pendant -SNO groups to come closer to each other.
Although the rate of decomposition of RSNO (or rate of
release of NO) is affected by heat, light, and metal ions as
discussed above, the major rate limiting step seems to be
the slower rate of diffusion (or reduced mobility) of the bulky
PVMMA-GS• or PVMMA-GSOO• radical and its recom-
bination with its parent molecule to release the NO and
forming disulfide linkages as described in the previous
section. As nitric oxide is gradually liberated from such a
complex, more disulfide bonds will form giving rise to in
situ disulfide cross-linking between RSNO side chains, which
(55) Picquart, M.; Grajcar, L.; Baron, M. H.; Abedinzadeh, Z.
Vibrational spectroscopic study of glutathione complexation in
aqueous solutions. Biospectroscopy 1999, 5, 328–337.
(56) Brandt, N. N.; Chikishev, A. Y.; Kargovsky, A. V.; Nazarov,
M. M.; Parashchuk, O. D.; Sapozhnikov, D. A.; Smirnova, I. N.;
Shkurinov, A. P.; Sumbatyan, N. V. Terahertz time-domain and
raman spectroscopy of the sulfur-containing peptide dimers: low-
frequency markers of disulfide bridges. Vib. Spectrosc. 2008, 47,
53–58.
(54) Stasko, N. A.; Fischer, T. H.; Schoenfisch, M. H. S-Nitrosothiol-
modified dendrimers as nitric oxide delivery vehicles. Biomac-
romolecules 2008, 9, 834–841.
(57) Grossi, L.; Montevecchi, P. C. A kinetic study of S-nitrosothiol
decomposition. Chem. Eur. J. 2002, 8, 380–387.
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Figure 8. Decomposition kinetics of (a) S-nitrosoPC5-
PVMMA/PVP (1/1) complex, (b) GSNO, and (c)
S-nitrosoPC5 in the presence of 36 µM [Cu2+] in 0.1 M
PBS (pH 7.4).
Figure 7. Comparison of Raman spectra of GSNO-
PVMMA/PVP (1/1) complex (a) before and (b) after NO
release.
presence of CuSO4 in an aqueous solution has been evaluated
and compared. The rate-determining step in this metal ion
catalyzed decomposition process is known to be the reaction
between RSNO and Cu+ (eq 8), where Cu+ can be generated
by the thiolate reduction of Cu2+ (eq 7) and has been deemed
to be the true catalyst.51,58
2RS- + 2Cu2+ f RSSR + 2Cu+
(7)
(8)
RSNO + Cu+ f RS- + Cu2+ + NO
The stability data of GSNO, S-nitrosoPC5 and S-nitroso-
PC5-PVMMA/PVP complex (with 3.24 wt % S-nitrosoPC5
loading) in phosphate buffer in the presence of copper ions
are shown in Figure 8. It can be seen that GSNO exhibits a
rapid degradation during the initial 20 min followed by a
progressively halted decomposition (curve b in Figure 8).
This seemingly incomplete degradation of GSNO through
the copper ion pathway can be attributed to the known
chelation reaction of Cu2+ with the GSSG already formed
in solution,59 which leads to less Cu2+ being available for
reduction. It is believed that the disulfide bridges pull together
the glutamate residues, facilitating the coordination of Cu2+
with sulfur or nitrogen atoms.51
Figure 9. Macroscopic evaluation of wound closure in
diabetic rats in GSNO-PVMMA/PVP (1/1) complex
treated group (n ) 7) versus the control group (n ) 5),
where * indicates p < 0.05.
starting concentration of -SNO (curve c in Figure 8). This
may be attributed to different complexation modes, as well
as different Cu2+:RSSR stoichiometries involving S-nitroso-
PC5.
On the other hand, S-nitrosoPC5-PVMMA/PVP shows
a substantially enhanced stability over the other RSNOs
(curve a in Figure 8); degradation of the complex is
negligibly slow under the same reaction conditions during
the initial 300 min. This is most likely due to the chelating
In contrast to GSNO, S-nitrosoPC5 shows linear decom-
position kinetics in the presence of copper from the same
(58) Noble, D. R.; Williams, D. L. H. Structure-reactivity studies of
the Cu2+-catalyzed decomposition of four S-nitrosothiols based
around the S-nitrosocysteine/S-nitrosoglutathione structures. Nitric
Oxide 2000, 4, 392–398.
(59) Hogg, N. Biological chemistry and clinical potential of S-
nitrosothiols. Free Radical Biol. Med. 2000, 28, 1478–1486.
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Figure 10. Representative photographs showing progression of wound healing in diabetic rats in GSNO-PVMMA/PVP
(1/1) complex treated group (T9) versus the control group (C3) at days 0, 4, 10, and 16 after wounding. The ruler
scale is 8 cm in all images. The integrated wound area is indicated in each photograph.
3). Representative photographs of full thickness wounds
showing the progression of wound healing in the test group
versus the control on days 0, 4, 10, and 16 are presented in
Figure 10. The apparent overall wound condition in terms
of wound closure and healing also appears to be better in
the test group than in the control group on days 4, 10, and
16 after wounding. These preliminary results strongly suggest
that the present controlled release NO delivery systems based
on GSNO-PVMMA/PVP interpolymer complexes could be
potentially useful in enhancing wound healing.
effect of the carboxylic acid polymer derived from hydrolysis
of the polyanhydride component (PVMMA) in the buffer.
This way, most of the Cu2+ may be removed from the
solution and not available to catalyze the decomposition, thus
resulting in enhanced stability.
Stability in the Solid State. As described above, the
conjugation of RSNO to PVMMA increases the bulkiness
of RSNO. Additionally, the physical interaction of PVMMA
and PVP in the complex effectively limits the mobility of
RSNO. Thus, the restricted mobility of macromolecular
radicals (PVMMA-GS•), due in part to the glassiness of
the physically cross-linked network in the solid state, is
expected to reduce the rate of decomposition by the collision
process. As a result, the present complex system remarkably
enhances the stability of RSNO in the solid state. There is
no significant change in the NO release profile after 6 months
storage or prolonged exposure to UV irradiation of the
GSNO-PVMMA/PVP complex powder under ambient
conditions (data not shown). This is indicative of the stability
of the present complex system in the solid state when stored
under room conditions.
Animal Studies. Wound closure data based on a diabetic
rat model are plotted in Figure 9, which demonstrate that
topical application of the present NO-releasing GSNO-
PVMMA/PVP (1:1) complex system can effectively acceler-
ate wound closure. There is a statistically significant differ-
ence in wound closure tendency between the control and test
group (p < 0.05) on days 4, 7 and 10, whereas the difference
becomes less significant on days 13 and 16. This may be
related to the fact that the NO release from the present
complex generally lasts only up to 10 days or so (see Figure
Conclusions
In conclusion, we have developed a novel but stable and
biocompatible platform for generating a durable release of
NO. We have reported the facile synthesis of new phytoch-
elatin-derived NO precursors (S-nitrosophytochelatins or
S-nitrosoPCs) and the formation for the first time of a novel
series of polymeric NO donors based on interpolymer
complexes between RSNO (GSNO and S-nitrosoPCs) con-
jugated PVMMA and poly(vinyl pyrrolidone) (PVP) through
intermolecular hydrogen bonding. In this case, the im-
mobilized NO precursors (RSNOs) are further stabilized in
a physically cross-linked polymer network. As a result, the
present interpolymer complex system exhibits remarkable
stability enhancement of immobilized GSNO in the solid
state as well as in the release medium due to significantly
reduced mobility of the resulting macromolecular glutathione
radicals leading to reduced rate of decomposition compared
with that of the free GSNO. In addition, the present complex
system also exhibits a substantial enhancement of stability
in the presence of copper ion in the release medium. The
existence of intermolecular hydrogen bonding in the present
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articles
Li and Lee
system and the formation of disulfide bonds as a result of
the NO release have been confirmed by FTIR and Raman
spectroscopy, respectively. We have also shown that the
present system is capable of providing sustained and
controlled release of NO for an extended duration of up to
10 days or more. Preliminary in ViVo testing in a diabetic
rat model demonstrates that topical application of the present
controlled release NO delivery system can effectively ac-
celerate wound closure. There is a statistically significant
difference in wound closure tendency between the control
and test group during NO release (p < 0.05). Since all
functional components of the present system have had prior
history of use in humans as well as in approved drug
products, this novel nitric oxide releasing platform can be
readily incorporated into dressings, patches and bandages
for diabetic wound treatment. Further studies to determine
the range of NO delivery rate suitable for diabetic wound
healing and to demonstrate enhanced wound healing quality
through histological assessment are in progress.
Acknowledgment. This work was supported by fund-
ing from the Natural Sciences and Engineering Research
Council of Canada (NSERC).
MP900237F
266 MOLECULAR PHARMACEUTICS VOL. 7, NO. 1