Clinical Science (2001) 100, 395–400 (Printed in Great Britain)
A novel method for the delivery of nitric oxide
therapy to the skin of human subjects using
a semi-permeable membrane
J. B. J. HARDWICK*, A. T. TUCKER†, M. WILKS‡, A. JOHNSTON* and N. BENJAMIN*
*Department of Clinical Pharmacology, St. Bartholomew’s and the Royal London School of Medicine and Dentistry,
Charterhouse Square, London EC1M 6BQ, U.K., †The Ernest Cooke Clinical Microvascular Unit, St. Bartholomew’s Hospital,
London EC1A 7BE, U.K., and ‡Department of Medical Microbiology, St. Bartholomew’s Hospital, London EC1A 7BE, U.K.
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Nitric oxide (NO) is a mediator of essential biological processes, including vasodilatation, antimicrobial activity and wound healing. A chemical system using sodium nitrite and ascorbic acid
has been developed which generates significant amounts of NO. The originally described system
was messy and impractical, and the high acidity may cause pain and further tissue damage in
ulcerated skin. To overcome this, a selectively permeable, hydrophilic polyester co-polymer
membrane system (Sympatex4) has been identified that can be placed between the NOgenerating chemicals and the skin. The aim of the present study was to determine whether NO
derived from this chemical system was able to diffuse through the membrane and have a
measurable vasodilatory effect on forearm skin in healthy volunteers. The Sympatex4 10 µm
membrane was found to be highly permeable to NO, while preventing passage of the constituents
of the NO-generation gel to the skin. The transmembrane NO-generation system had a
vasodilatory effect comparable with that resulting from direct topical application. Additionally,
the NO generated was effective in killing Staphylococcus aureus and Escherichia coli at doses lower
than those required to increase skin blood flow. The vasodilatory and anti-microbial effects of
this system may be useful as a patch-based topical therapy for skin ulceration, particularly when
there is concomitant ischaemia and infection.
INTRODUCTION
Nitric oxide (NO) is generated from L-arginine via NO
synthase enzymes and performs a variety of functions,
including vasodilatation and host defence [1]. We have
shown that NO is also manufactured on epithelial
surfaces (such as in the mouth and stomach, and on the
skin surface) in humans by sequential reduction of nitrate
and nitrite [2]. This relies on the synthesis of nitrite by the
bacterial reduction of inorganic nitrate present in saliva
or sweat. Nitrite is further reduced to NO in an acidic
environment, a reaction which is particularly enhanced in
the presence of ascorbic acid :
NO −jH+
HNO
#
#
2HNO
N O jH
3HNO
2NOjNO −jH+jH O
#
#
# $
NOjNO
#
#
$
#
(1)
The combination of acid and nitrite is effective in
killing a wide variety of pathogens [3,4], presumably by
the generation of nitrogen oxides. It is likely that NO
generated in this way has a significant role in host defence
Key words : anti-microbial, blood flow, diffusion, infection, laser Doppler, nitric oxide, skin.
Abbreviations : LDF, laser Doppler fluxmetry.
Correspondence : Dr A. T. Tucker (e-mail a.t.tucker!mds.qmw.ac.uk).
# 2001 The Biochemical Society and the Medical Research Society
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J. B. J. Hardwick and others
against microbial pathogens, many of which are known
to be susceptible to this agent [5].
We have devised a system which mimics this endogenous mechanism of NO generation, using inorganic
nitrite and an organic acid to produce NO on the skin
surface. This method relies on keeping the components
separate until applied directly to the skin. When used in
this way, the system is effective in treating infectious skin
disease [4] and increasing skin blood flow [6]. Individually, these components elicit no significant effects.
However, the previously described direct contact
application was messy and impractical. In addition, we
have noted previously in three patients with digital
ulceration that the acidic nature of this NO-generating
system causes pain and may cause further damage. To
resolve these adverse aspects, we have devised a system
intended to protect the skin from the acid contained in
the nitrite\acid mixture, but which still allows the known
actions of the NO generated, such as anti-microbial
effects and vasodilatation. This involves the use of a
selectively permeable, hydrophilic, polyester co-polymer
membrane (Sympatex4 10 µm membrane ; Azko Nobel,
Wuppertal, Germany) to separate the acidic mixture
from the tissue ; this membrane still allows NO to pass
freely into the skin.
Studies were designed to determine the efficacy of this
novel NO delivery system using in vivo studies of human
forearm to measure skin blood flow, and in vitro studies
of the anti-microbial action of NO delivered through a
gas-permeable membrane system. The topical application
of such a system may be a useful therapy for skin
ulceration or chronic wound infection, as it has been
shown to be important in promoting effective wound
healing [7–9] and exhibits potent anti-microbial properties [3,5].
METHODS
Measurement of NO diffusion across gaspermeable membranes
Nitric oxide was generated using sodium nitrite and
ascorbic acid (eqn 1). The experimental arrangement is
shown in Figure 1.
The constitutive ingredients were made up separately
in 0n8 % (w\v) agar. The Sympatex4 10 µm membrane
was placed over the sampling area of 1n33 cm#, on to
which 1 ml of each mixture was placed. The two agents
were combined gently using a sterile swab stick, and the
rate of NO diffusion was measured by passing air, free of
NO and nitrogen dioxide, over the other side of the
Sympatex4 and into the chemiluminescence NO meter.
The rate of NO generation was measured by a sensitive
chemiluminescence meter (model 42C ; Thermo Environmental Instruments Inc., MA, U.S.A.) connected to a
data acquisition system and IBM computer, over a 20 h
# 2001 The Biochemical Society and the Medical Research Society
Figure 1 Diagram showing the experimental arrangement
of the chemiluminscence NO/NO2/NOx sampling chamber
The NO generation gel was mixed in the sealed upper reaction chamber. NO that
diffused into the lower collection chamber was aspirated continuously, and the NO
concentration was measured. Comparisons were made between conditions of free
diffusion and the situation when the chambers were separated and sealed by the
Sympatex4 membrane.
period. The gas phase in the lower collection chamber
was sampled every 60 s.
The rate of release of NO from the gel surface
(measured as nmol:s−":cm−#) for 330 mM concentrations
of sodium nitrite and ascorbic acid was measured, and
compared with the rate of passage of NO through the
Sympatex4 membrane.
Skin blood flow experiment
The protocol was approved by the East London and City
Health Authority Ethics Committee, and subjects gave
written informed consent prior to participation in the
study, which was carried out according to the Declaration
of Helsinki. The study group consisted of nine healthy
male volunteers aged between 20 and 30 years. The
subjects’ demographic data are summarized in Table 1.
Subjects were non-smokers, normotensive, normocholesterolaemic and normoglycaemic, with no evidence
or history of auto-immune or vascular disease, and were
not taking any medication.
All examinations were performed in a draught-free,
temperature- and humidity-controlled laboratory
(24p1 mC ; relative humidity 30–40 %) over a 2-week
period. All subjects had a light breakfast, avoiding fatty
foods, tobacco consumption and caffeine, and had
abstained from vigorous exercise since the previous
evening. The microvascular measurement techniques
were non-invasive and the investigation was consistently
reported to be painless. The volunteer was required to sit,
lightly clad, with their arms supinated, slightly bent and
supported by a foam stand at an angle of 30m to the
horizontal.
Assessment of microvascular peripheral blood flow in
the arm used laser Doppler fluxmetry (LDF) (model
Transdermal nitric oxide therapy
Table 1
Demographic summary (n l 9)
Parameter
Mean
S.E.M.
Median
Age (years)
Body mass index (kg/m2)
Systolic blood pressure (mmHg)
Diastolic blood pressure (mmHg)
Heart rate (beats/min)
25n6
25n3
117n4
78n9
74n0
0n9
1n2
2n4
1n5
1n6
25
24
118
78
74
DRT-4 laser Doppler fluxmeter ; Moor Instruments
Ltd, Axminster, Devon, U.K.). LDF is a technique that
uses the Doppler shift of laser light of wavelength
780–810 nm to measure the flux (velocityinumber) of
red blood cells. IR light generated by the laser is directed
to the tissue via an optical fibre ; back-scattered light
reflected from moving whole blood particles is then
measured by one or more optical fibres [10].
The surface of one forearm was covered with
Sympatex4 ; the other arm was in direct contact with the
NO-generating gel. Selection of the arm that was to be in
direct contact with the gel was determined by a sequence
of random numbers. An integrated LDF probe was
placed on the centre of the flexor surface of each forearm,
with the centre equidistant from the lateral and medial
borders, and marked on the skin to ensure accurate repositioning. Baseline measurements were taken for 3 min,
after which 1 ml each of sodium nitrite (330 mM) and
ascorbic acid (330 mM) was added to the surface of each
arm (either directly on to the skin or on to the
Sympatex4) and mixed to initiate NO generation (see
eqn 1). Measurements of skin laser Doppler flux were
then taken continuously for a further 50 min.
Preliminary studies have shown that KY jelly4 containing sodium nitrite alone causes vasodilatation in the
forearm skin of healthy volunteers ; however, this is not a
statistically significant increase and is not sustained.
Additionally, KY jelly4 containing ascorbic acid has no
affect on skin microcirculation.
Statistical comparison between treatments was carried
out using ANOVA.
Assessment of transmembrane antimicrobial activity
Nitric oxide was generated by the system described in the
Introduction. The anti-microbial effects of NO were
tested using Staphylococcus aureus NCTC9353 and
Escherichia coli NCTC10148 using a range of concentrations of sodium nitrite and ascorbic acid (1000, 500,
330, 100, 50, 10, 5 and 1 mM).
A sample of 1 ml of an overnight culture of E. coli or S.
aureus (mean counts of 8n8i10% and 4n95i10% colonyforming units respectively) was inoculated into 24 ml of
1n5 % (w\v) agar in 1 % (w\v) NaCl at 45 mC, which was
then allowed to set. Discs of Sympatex4 membrane
Figure 2 Diagram showing the experimental arrangement
of the Sympatex4 membrane and the inverted inoculated
agar
(9 cm in diameter) were sterilized in 70 % (v\v) ethanol
and left to dry by evaporation in a laminar flow cabinet.
Samples of 5 ml of 0n8 % agar in 1 % NaCl, containing
sodium nitrite or ascorbic acid at concentrations of 1000,
500, 330, 100, 50, 10, 5 or 1 mM, were prepared.
In the centre of inverted Petri dish lids (sterile), 1 ml
samples of each concentration of sodium nitrite and
ascorbic acid were added and mixed. Disinfected
Sympatex4 membrane was placed over the top of this
immediately using sterilized forceps, ensuring that it
overhung the lid in all directions. The base of the Petri
dish, containing the set inoculated agar, was placed upside
down on top of the lid\mixture\Sympatex4 membrane
arrangement, ensuring that a 2–3 mm gap was left
between the Sympatex4 membrane and the inverted
inoculated agar (Figure 2).
The apparatus was incubated overnight at 37 mC. The
base of the Petri dish was then removed and the central
area of agar was sampled by cutting a circle using a sterile
plastic measuring cup. The agar was then homogenized
by vortexing with sterile glass beads (1 mm diameter) for
4 min. Next, 10-fold serial dilutions were carried out, and
the samples of each dilution were plated on to blood\agar
plates. After incubation for 24 h at 37 mC, the resulting
colony-forming units were then counted and percentage
survival calculated.
RESULTS
Permeability of Sympatex4 to NO
The Sympatex4 10 µm membrane was found to be
highly permeable to NO (diffusion coefficient l
0n005i10−' cm:s−"), while preventing passage of the
# 2001 The Biochemical Society and the Medical Research Society
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J. B. J. Hardwick and others
Figure 3
to NO
Permeability of the Sympatex4 10 µm membrane
The concentration of sodium nitrite and ascorbic acid was 330 mM. PPM, parts per
million.
Figure 5 Survival of E. coli and S. aureus after 24 h of
exposure to NO diffused through a Sympatex4 10 µm
membrane
=, E. coli (NCTC10148) ; #, S. aureus (NCTC9353). Values are medians
(n l 3).
10 µm membrane are shown in Figure 4. The results of
baseline forearm microcirculatory blood flow measurements from each arm were not statistically different
(mean 4n19 flux units ; 95 % confidence interval 4n00–5n30
units). Following application of the NO-generating
system, forearm LDF measurements showed a significant
increase in microcirculatory blood flow from baseline
(P 0n0001 ; ANOVA). The mean vasodilatation measured by LDF in the presence of the Sympatex4 10 µm
membrane was 64 % of that observed when the NOreleasing gel was applied directly to the skin.
Figure 4 Forearm microvascular flux responses to application of the NO generation gel in the presence and absence
of a Sympatex4 10 µm membrane
constituents of the NO-generation gel to the skin.
Although a variety of plastics and membranes are
partially permeable to NO, preliminary studies had
shown that all were less than half as permeable as the
Sympatex4, and in addition did not restrict passage of
the gel components effectively.
Compared with the direct production of NO, the peak
liberation of NO through the Sympatex4 10 µm membrane was lowered by 50 %, and the time taken to reach
the peak value was extended by 4 min to 11 min. The
kinetics of NO diffusion are shown in Figure 3.
Effect on microcirculatory blood flow
The effects of a single dose (330 mM) of nitrite and
ascorbic acid applied topically and through a Sympatex4
# 2001 The Biochemical Society and the Medical Research Society
Transmembrane anti-microbial effect
Potent anti-microbial properties of NO were seen at
concentrations of nitrite above 50 mM, resulting in
complete killing of bacteria to below detectable levels. At
lower concentrations, partial or no anti-microbial activity
was seen, which was dose-dependent (Figure 5).
DISCUSSION
Chronic leg ulceration and wound infections are common
disorders, which are placing an increasing strain on health
service providers [11]. Chronicity is due primarily to the
nature of the underlying causes of colonization with
infective organisms and of local tissue ischaemia seen in
diabetic subjects and in patients with peripheral vascular
disease.
Current treatment regimens used for wound infection
and leg ulceration include the administration of anti-
Transdermal nitric oxide therapy
biotics, which may in time lead to the evolution of
antibiotic-resistant strains of the organism, and the
application of compression bandages as appropriate.
Pathological changes in the microcirculation associated
with ulceration are not addressed by current available
treatments, which tend to rely on stimulating granulation
of the wound [12].
With respect to this current problem in the management of wound infections and skin ulceration, and in
view of the knowledge that NO has been shown to (1)
increase microcirculatory blood flow [8], (2) kill a range
of infective organisms [4,5,13], and (3) have a significant
effect in promoting effective wound healing [7–9], we
propose that the NO delivery system described in the
present paper may be used in the treatment of such
disorders.
The present study demonstrates an effective method
for the delivery of NO to the skin surface using acidified
nitrite. The system delivers sufficient NO to cause
significant vasodilatation in vivo and to kill S. aureus and
E. coli in vitro. An additional advantage of the system is
that the potentially damaging acidic solution is prevented
from coming into contact with the skin surface by the use
of a Sympatex4 membrane, which may be developed into
a self-contained delivery patch system.
The increase in microcirculatory blood flow seen in the
arms covered with the Sympatex4 membrane confirms
previous findings that NO has a vasodilatory effect [6].
The suggested mechanism of vasodilatation is that NO is
able to diffuse 1–3 mm through the skin to the precapillary sphincters without being oxidized [10].
The baseline microcirculatory blood flow measurements in the two forearms were not significantly different. However, an increase in skin blood flow was
noticeable almost immediately after the gels were applied,
was maintained throughout the experiment and was
statistically significant. The arm to which the NOgeneration system was incorporated with the Sympatex4
membrane showed less of an increase in microcirculatory
blood flow. These results are due to the membrane
resisting the passage of the generated NO, therefore
increasing the amount escaping to the atmosphere.
Additionally, the Sympatex4 membrane may have
altered the optical properties of the laser Doppler
fluxmeter. It is evident from Figure 2 that the initial rate
of increase was greater following the direct application of
the NO-generating system. This finding demonstrates
that the Sympatex4 membrane shows some resistance to
the passage of NO.
The amount of NO released from this system is capable
of initiating complete killing of two important pathogens,
S. aureus and E. coli. Previous studies using acidified
nitrite have shown that this combination is effective in
killing a wide range of human pathogens, including Tinea
pedis [4] and E. coli [5]. Reactive nitrogen intermediates
are involved in the killing of S. aureus in human blood
neutrophils [14]. The mechanism behind this NOmediated killing is thought to involve oxygen and\or
oxygen free radicals, which the NO encounters, leading
to the production of nitrogen radicals [12]. The antimicrobial action of NO could also be attributed to the
ease with which NO crosses cell membranes and inhibits
respiratory chain enzymes via activation of sulphur–iron
complexes [5].
These findings suggest that development of this NOgenerating system may be particularly useful in the
management of skin infections where there is ulceration
and when exposure to acid should be avoided. The
obvious clinical problem which may benefit from both
increased skin blood flow and anti-microbial action is
that of leg and foot ulcers, which are commonly resistant
to conventional therapies.
Inhibition of NO synthase, the enzyme that mediates
the production of NO from the amino acid L-arginine [1],
impairs wound healing. NO may have benefits in the
promotion of wound healing post-operatively, as it
promotes angiogenesis, cell proliferation and migration,
and anti-platelet activity [7]. Additionally, urinary nitrate
excretion is elevated after incisional wounding [15] and in
burns [16]. The topical application of the NO-generating
gel and membrane may be of benefit to patients with leg
ulceration and wound infection. It may also promote
post-operative wound healing, and help to prevent
wound infection in patients with burns and those with
skin grafts.
Further planned studies involve obtaining mixed
microbiological cultures from infected wounds and leg
ulcers and exposing them to NO generated by the
improved system. Additionally, the increase in microcirculatory blood flow may not necessarily equate to
increased tissue oxygenation, particularly in areas of
ulceration [17,18]. This is an area needing further
investigation, with the use of a transcutaneous O and
#
CO meter. The development of a patch system, allowing
#
the ascorbic acid and sodium nitrite gels to mix when
applied, would facilitate usage in a clinical environment
and give patients the ability to self-administer. Another
advantage of such a patch system would be to maximize
the concentration of NO delivered locally, both
prolonging and increasing anti-microbial and vasodilatory activities.
In summary, we have described a novel NO generation
and delivery system. The significant development of this
system over previous reports has been the identification
and inclusion of a selectively permeable membrane,
which allows the diffusion of the NO generated towards
the skin. This delivery of NO to the skin significantly
increases skin blood flow in healthy volunteers and
completely lyses S. aureus and E. coli in vivo. The system
may therefore be beneficial in the promotion of healing in
skin ulceration, wound infection, burn injuries and skin
grafts.
# 2001 The Biochemical Society and the Medical Research Society
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J. B. J. Hardwick and others
ACKNOWLEDGMENTS
We thank Sympatex Technologies G.m.b.H. (Wuppertal,
Germany) for the supply of the membrane for this study.
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Received 6 July 2000/16 November 2000; accepted 14 December 2000
# 2001 The Biochemical Society and the Medical Research Society