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Stability Study of Novel Medicated Hydrogel Wound

Dressings
a a a
Niladri Roy , Nabanita Saha & Petr Saha
a
Centre of Polymer Systems , Tomas Bata University in Zlin , Zlin , Czech Republic
Accepted author version posted online: 06 Jun 2012.Published online: 14 Jan 2013.

To cite this article: Niladri Roy , Nabanita Saha & Petr Saha (2013) Stability Study of Novel Medicated Hydrogel
Wound Dressings, International Journal of Polymeric Materials and Polymeric Biomaterials, 62:3, 150-156, DOI:
10.1080/00914037.2011.641697

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 International Journal of Polymeric Materials, 62: 150–156
Copyright # 2013 Taylor & Francis Group, LLC
ISSN: 0091-4037 print/1563-535X online
DOI: 10.1080/00914037.2011.641697

Stability Study of Novel Medicated Hydrogel Wound

Dressings
NILADRI ROY, NABANITA SAHA, and PETR SAHA
Centre of Polymer Systems, Tomas Bata University in Zlin, Zlin, Czech Republic

Received 7 June 2011, Accepted 10 November 2011

Downloaded by [Indian Institute of Technology - Kharagpur] at 22:51 29 April 2015

PVP-CMC and PVP-CMC-BA hydrogels were prepared for biomedical applications. These hydrogels were stored under
5
C  2
C, 22
C  3
C, and 40
C for 180 days to identify their suitable storage condition. At 5
C  2
C, hydrogels showed mini-
mum alteration in physical properties. No microbial colonies appeared on PVP-CMC stored at 5
C  2
C, but they slowly
appeared at 22
C  3
C and 40
C after 45 days. FTIR and antibacterial assay demonstrated that boric acid (BA) in hydrogel
remained stable for 180 days. PVP-CMC-BA is more bio-stable than PVP-CMC because of BA, an antibacterial agent. Finally,
5
C  2
C was recommended as the best storage condition for both PVP-CMC and PVP-CMC-BA hydrogels.
Keywords: Biomaterials, boric acid, hydrogels, stability, wound dressing

1. Introduction
The concept of hydrogels used in medical applications is not
new. Hydrogels were first introduced for clinical purposes in
ophthalmological use in the early 1950s by Wichterle and
Lim [1]. Hydrogels have various applications in biomedicine
[2–6]; among them, hydrogels for wound dressing have one
of the major implementations in this domain. Hydrogel
use for wound dressing was invented in the late 1980s by
Rosiak et al. [7,8]. Hydrogels can be successfully used as
dressing materials in medical treatment for burns and
wounds because a wet environment can enhance the wound-
healing process [9–12]. Due to their high water content,
hydrogels possess excellent biocompatibility and a degree
of flexibility similar to natural tissues, minimizing potential
irritation to surrounding membranes and tissues [13–16].
Thus, the main goal of wound-dressing hydrogels is for
application on the patients’ skin lesions [17]. It should be
noted that medical device use has expiration dates, and use
needs to take place before those dates. After that period, it
can be dangerous and may cause harm. The adverse effect
can be mild to severe. Therefore, when hydrogels are con-
sidered for use in medical devices, it is necessary to know
their durability for safe use. The durability highly depends
on the environmental conditions under which the material
was stored. It is very important to know the appropriate
storage conditions which are suitable for the longer stability
of the materials. The objective of a stability study is to deter-
mine the shelf-life, namely the time period of storage at a
Address correspondence to: Nabanita Saha, Polymer Centre,
Centre of Polymer Systems, Tomas Bata University in Zlin,
Nam T. G. Masaryka 5555, 76001 Zlin, Czech Republic.
E-mail: nabanitas@yahoo.com; nabanita@ft.utb.cz
specified condition within which the product still meets its
established specifications [18]. Stability is an essential factor
to maintain the quality, safety, and efficiency of a medical
device. A product that is not of a sufficient stability can
result in changes in physical (hardness, dissolution rate,
phase separation, etc.) as well as chemical characteristics
(formation of high-risk decomposition substances). Micro-
biological instability of a sterile product could also be
hazardous [18]. The stability study consists of a series of tests
in order to obtain an assurance of stability of the product
packed in its specified packaging material and stored in the
established storage condition within the determined time
period. Hydrogels were prepared in combination with syn-
thetic and natural polymers like polyvinylpyrrolidone
(PVP) and carboxymethyl cellulose (CMC), along with other
ingredients, and boric acid (BA) was incorporated within the
hydrogel to acquire antimicrobial property within it.
Further, it has been established that both PVP-CMC and
PVP-CMC-BA hydrogels are ideal for wound dressings
and recommended for their safe use in humans=animals after
thorough examination and characterization of such aspects
as swelling; moisture content; moisture-retention capacity;
ability for water vapor transmission; internal and external
morphology; physico-chemical, mechanical, viscoelastic,
and antimicrobial properties; microbial penetrability and
biocompatibility with respect to cytotoxicity; skin irritation
and skin sensitization tests, etc. [19–26]. Finally, it is impor-
tant to examine the storage condition of the hydrogels
during product delivery. Moreover, it is essential to preserve
the valuable properties of hydrogels specifically prepared for
health-care purpose. Thus, special attention needs to be
given to their biological and environmental stability. The
aim of this study is to identify the durability or stability of
the hydrogels under different storage temperatures and to
 Stability Study of Hydrogel Wound Dressings
find out the appropriate storage condition for the novel
hydrogel wound dressings. The present paper focuses on
the occurrence of changes observed in PVP-CMC and
PVP-CMC-BA hydrogels at different storage temperatures
on the basis of their significant existing properties: physical
appearance (morphology, thickness, size, texture, color);
moisture content; preservation of sterility; and retention of
boric acid activity (i.e., on the basis of antibacterial effective-
ness), etc.
2. Experimental
2.1 Materials for Hydrogel Preparation
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Polyvinylpyrrolidone K 30 (PVP: molecular weight 40,000),
polyethylene glycol 3,000 (PEG: average molecular weight
3,015-3,685), and agar were obtained from Fluka, Switzer-
land; sodium carboxymethyl cellulose (CMC) was purchased
from Sinopharm Chem. Reagent Co., Ltd, China; boric acid
(BA) from Sigma-Aldrich, USA; and glycerin obtained from
Lachema Ltd, Czech Republic.
2.2 Preparation of Hydrogels
The ingredients used for the preparation of hydrogels were
PVP, CMC, BA, PEG, agar, and glycerin. The polymer solu-
tions were prepared in sealed bottles by dissolving weighed
components in water. Then, moist heat treatment [15 lbs
(107 kPa) pressure, 120
C, 20 minutes] [19,20] was applied
to the solutions, followed by solution (5 ml each) casting in
molds of 50 mm diameter. The polymer solutions were
allowed to cool at room temperature (22
C  3
C) under
an aseptic environment to achieve the desired hydrogels.
The hydrogel without BA was designated ‘‘PVP-CMC’’
and the hydrogels with BA as ‘‘PVP-CMC-BA.’’ The initial
composition of each test samples is given in Table 1.
2.3 Experimental Parameter/Testing Methods
To identify the suitable storage conditions for PVP-CMC
and PVP-CMC-BA hydrogels, some selected tests such as
retention of moisture content, changes in physical appear-
ance=behavior, preservation of sterility, retention of BA
activity, etc., were carried out and are mentioned below.
2.4 Measurement of Moisture Content [19,27]
Moisture content of the hydrogels was measured gravimetri-
cally using Equation (1) in which Mn ¼ moisture content (%)
of material n, WW ¼ wet weight of the sample, and
Wd ¼ weight of the sample after drying. The wet weight of
Table 1. Composition of hydrogels (W=V %) [20 and 23]
Sample
Index PVP CMC PEG Agar Glycerin BA Water
PVP-CMC 0.2 0.8 1 2 1 0 95
PVP-CMC- 0.2 0.8 1 2 1 3 92
BA
151
the hydrogels was recorded after each specific time interval
for the samples from each storage condition and then the
samples were dried under room temperature until they
assumed constant weight and then the measurements were
done. In each case three replicates were taken and the
average value of moisture content was determined.
ðMn Þ% ¼ ½ðWw  Wd Þ=Ww   100 ð1Þ
2.5 Physical Appearance
Changes in physical appearance of the material can arise due
to the deviation from the established specifications; i.e., it
refers to the fact that the material is not more stable and
reached the end of its shelf-life. Three parameters were
checked periodically for 180 days: (a) Weight of the samples
was measured by analytical balance and data was recorded;
(b) size and thickness of the hydrogels were measured using a
ruler and micrometer; (c) color, texture, and appearance
were observed visually and any differences were noted.
2.6 Preservation of Sterility
Maintenance of the sterility is one of the most important
factors for medical devices. As long as the sterility of the bio-
material persists, no microbe can grow on the hydrogel.
Thus, to confirm the stability of the hydrogel from a biologi-
cal point of view, the hydrogels were checked time to time
with careful visual observation, i.e., appearance of any
microbial colonies on the hydrogel.
2.7 Retention of Boric Acid Activity in PVP-CMC-BA
Hydrogel
BA was incorporated in the hydrogel to achieve medicinal
value and antimicrobial property within the hydrogel. The
dose of BA in the hydrogel was maintained at 3% as it is
the permissible limit of BA for health-care purposes
[20,28]. It is essential to check the persistence of BA in stable
form as BA is the responsible ingredient to preserve the anti-
microbial property of hydrogel. If BA degrades with time, it
will lose its antimicrobial activity and destabilize the hydro-
gel. The retention of BA activity was assayed on the basis of
FTIR measurement as well as on the basis of antibacterial
effectiveness (measuring the zone of inhibition).
2.7.1 FTIR of PVP-CMC-BA Hydrogel
PVP-CMC-BA hydrogels in dry form were analyzed by
FTIR. The ATR-FTIR spectroscopic analysis was conduc-
ted using a NICOLET 320 FTIR Spectrophotometer with
‘‘Omnic’’ software package over the range 4000–600 cm1
at room temperature. A uniform resolution of 2 cm1 was
maintained in all cases. The FTIR spectra values between
1800–1000 cm1 have been considered to evaluate the
stability of BA.
2.7.2 Antimicrobial Assay of PVP-CMC-BA Hydrogel
The antimicrobial properties of PVP-CMC-BA hydrogels
were investigated by agar diffusion method [20,29–31]. Anti-
microbial characteristics of these hydrogels were examined
on the basis of the dimension of the inhibition zone
 152
generated in the presence of pathogenic bacteria like Escher-
ichia coli and Staphylococcus aureus. The antibacterial assay
was conducted in a sterile nutrient agar (3%) medium, which
was inoculated with the above-mentioned bacterial strains
under aseptic condition. After solidification of nutrient agar,
a piece of hydrogel (15 mm diameter) obtained from three
different storage conditions (5
C  2
C, 22
C  3
C, and
40
C) was placed on the surface of the medium. The testing
plates were then incubated in a temperature-controlled incu-
bator at 37
C to observe the effect of PVP-CMC-BA hydro-
gels on the growth of individual bacteria. The antimicrobial
assay was carried out with the PVP-CMC-BA hydrogel sam-
ple isolated from different temperatures (5
C  2
C, 22
C 
3
C, and 40
C) at different intervals (i.e., 0, 45, 90, 180
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days). The persistence of antibacterial activity of BA was
evaluated on the basis of zone of inhibition.
3. Results and Discussions
3.1 Moisture Content of Hydrogels
Moisture-holding capacity is one of the most important
properties for hydrogels. Because of it, hydrogels are able
to provide a moist environment to the wound; moreover, it
is proven that the moist environment helps to heal the
wound faster [9–12]. Thus, evaluation of the moisture-
retention capacity of both PVP-CMC and PVP-CMC-BA
under different storage conditions is important and is shown
in Figure 1. It can be seen from Figure 1 that under
5
C  2
C (in refrigerator) both the hydrogels lose very
nominal amounts of water up to 180 days of study. Even
under 22
C  3
C (room temperature), the hydrogels do
not show remarkable moisture loss. However, under 40
C,
the hydrogels lose water sharply and tend to dry with time.
After 180 days, all the PVP-CMC-BA samples were found to
be totally dry. At higher temperature (40
C), the water from
the hydrogels evaporates faster, which causes higher degree
of moisture loss from the hydrogels, thus becoming almost
or totally dry after 180 days.
Fig. 1. Effect of storage conditions on moisture content of
hydrogels. (Figure is provided in color online.)
N. Roy et al.
3.2 Physical Appearance of Hydrogels
Physical appearance of the materials offers lots of infor-
mation concerning the stability of hydrogels. Figure 2 shows
the optical view of the initial samples and the samples after
180 days of storage at different temperatures for PVP-CMC
and PVP-CMC-BA hydrogels. Initial samples of PVP-CMC,
when dried at room temperature, appear transparent, but in
the case of dry PVP-CMC-BA white dots of BA appear
throughout the film [20]. From Figure 2, it is noticeable that
after 180 days at 40
C the PVP-CMC-BA hydrogel becomes
totally dry and brittle, and BA comes out because of the effect
of the high temperature. The drying process of the hydrogels
become faster at higher temperature, and during drying water
evaporates from the hydrogels and, after a certain time, the
dissolved BA reaches its saturation and re-crystallizes, which
appears as white dots [20]. It has been observed that, in the
case of PVP-CMC hydrogel, until the end of the experiment
there was no significant color change, but in the case of
PVP-CMC-BA, at 40
C, from 90 days onwards white spots
of BA appeared and it were spread like a network throughout
the hydrogels. This appeared because, at higher temperature,
the hydrogels started to lose water and oversaturated BA
became visible. In the case of both hydrogels, the rate of
weight loss (Figure 3) and reduction of thickness (Figure 4)
are remarkably higher for the samples stored at 40
C; conse-
quently the same trend has been observed in the reduction of
the diameters of the hydrogels. The changes are moderate for
the samples at 22
C  3
C, but very little at 5
C  2
C
because of the variation in temperature. The rate of evapor-
ation of water from the hydrogels varies in the following man-
ner: 5
C  2
C < 22
C  3
C < 40
C.
3.3 Preservation of Sterility of Hydrogels
The appearance of microbial colonies on the material refers
to the fact that the material is no longer sterile, which means
it loses its biological stability. Thus, it is important to check
thoroughly the appearance of microbial colonies on the sur-
face of the hydrogels in programmed intervals, the results of
which are reported in Table 2. In the case of PVP-CMC
hydrogel at 22
C  3
C and 40
C, some microbial colonies
appeared around 45 days, as shown in Figure 5. The bac-
terial growth was visible within 75 to 90 days, which may
be due to the availability of suitable environmental con-
ditions for bacterial growth. After that, there was no sign
of bacterial colonies. The reason for the disappearance of
bacterial colonies may be due to the fact that the growth
of bacteria reached into a declining phase. It can also be
explained as follows: the microbes that appeared could not
survive for a long time due to the temperature, lack of oxy-
gen, and other environmental influences. The temperature of
5
C  2
C seemed to be a very suitable condition for storing
the PVP-CMC hydrogels, as there was no appearance of
microbial colonies for the 180 days of our investigation. In
the case of PVP-CMC-BA, under all the storage conditions
no microbial colonies appeared at all, because BA is an anti-
microbial agent and it resisted the hydrogels from any
microbial contamination.
 Stability Study of Hydrogel Wound Dressings 153
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Fig. 2. Optical image of the hydrogels at different storage conditions. (Figure is provided in color online.)

Fig. 3. Weight loss profiles of hydrogels with respect to time under different storage conditions: (a) PVP-CMC; (b) PVP-CMC-BA.
(Figure is provided in color online.)
 154 N. Roy et al.
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Fig. 5. A view of microbial colonies on the PVP-CMC hydrogel

(appeared after 45 days when sample was stored at 40
C).
(Figure is provided in color online.)

compare the stability of BA as well as other components

Fig. 4. Effect of storage conditions on the thickness of hydro-
gels: (a) PVP-CMC; (b) PVP-CMC-BA. (Figure is provided in
color online.)
3.4 Retention of Boric Acid Activity in PVP-CMC-BA
Hydrogel
3.4.1 FTIR of PVP-CMC-BA Hydrogel
The FTIR spectra of PVP-CMC-BA hydrogels (initial and the
samples after 180 days of storage) were studied in a dry state to
present in hydrogel, and the results are illustrated in Figure 6.
In our previous published work, [20] we had shown that BA
shows a characteristic broad peak just above 1400 cm1 and
another peak at 1189 cm1 in the case of PVP-CMC-BA. In
this study also all the samples [initial and the samples stored
for 180 days] show BA peaks at 1406 cm1 and 1189 cm1.
For all the samples, BA peak intensities appear very close to
each other. This behavior proves that, during 180 days of
storage, activity of BA in the PVP-CMC-BA hydrogel was
not affected considerably by different storage temperatures.
Another peak appears at 1632 cm1, because the 1,658 cm1
peak of PVP and 1,588 cm1 peak of CMC become a single
broad peak at 1632 cm1 [20]. The intensities of the peaks at
1632 cm1 of the PVP-CMC-BA hydrogel samples vary little
among themselves. Also, the 1280 cm1 peak of C-N stretching
vibration of PVP and 1062 cm1 peak of 1,4-b-D-glucosidic
stretching vibration of CMC [19,20] in initial PVP-CMC-BA
either diminish or shift for the samples after 180 days.
Therefore, it can be mentioned that the storage temperatures
do not affect the activity of BA throughout the experimental

Table 2. Antibacterial effectiveness of boric acid present in PVP-CMC-BA hydrogel

Effective diameter of inhibition zone Effective diameter of inhibition zone

Storage developed by PVP-CMC-BA in presence of developed by PVP-CMC-BA in presence of
condition of Staphylococcus aureus (mm) Escherichia coli (mm)
hydrogels
Room Temp controlled Temp controlled Room Temp controlled Temp controlled
temperature fridge incubator temperature fridge incubator
Days (22
C  3
C) (5
C  2
C) (40
C) (22
C  3
C) (5
C  2
C) (40
C)

0 20 20 20 5 5 5
45 20 20 20 5 5 5
90 20 20 18 5 4 4
180 19 18 9 Not clear Not clear Not clear
Temp ¼ Temperature.
 Stability Study of Hydrogel Wound Dressings
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Fig. 6. FTIR spectra of PVP-CMC-BA hydrogels (in dry form)
stored under different storage conditions. (Figure is provided in
color online.)
period, but it may affect up to a certain extent the properties of
base polymers like PVP and CMC, as they are biomaterials.
3.4.2 Antimicrobial Assay of PVP-CMC-BA Hydrogel
Boric acid (BA) was incorporated into the PVP-CMC hydro-
gel to achieve an antimicrobial-property-based hydrogel.
The PVP-CMC-BA hydrogel containing 3% BA shows very
good resistant property against microbes [20]. Thus, it is
necessary to investigate the effect of different temperatures
Fig. 7. Image of antibacterial effect of PVP-CMC-BA hydrogels in the presence of skin-infection-causing bacteria Escherichia coli
(a, c) and Staphylococcus aureus (b, d) [initial and sample after 180 days’ storage at (1) 22
C  3
C; (2) 5
C  2
C; (3) 40
C]. (Figure
is provided in color online.)
155
and time on the activity of BA from the stability point of
view of the hydrogels. The effect of storage conditions on
the antimicrobial effect of boric acid are presented in
Table 2, by means of development of the zone of inhibition
in the presence of pathogenic bacteria like Escherichia coli
(E. coli) and Staphylococcus aureus (S. aur). From the
observed data, it can be said that BA keeps up its antibacter-
ial activity throughout the experimental period (180 days).
Figure 7 shows the antibacterial effect of the PVP-CMC-
BA hydrogel (initially and after 180 days, sample stored
under different storage temperatures) in the presence of
skin-infection-causing bacteria (a) E. coli and (b) S. aur. It
can be seen from Figure 7 that the PVP-CMC-BA hydrogel
(stored under 40
C) shows smaller (in the presence of S. aur)
or unclear (in the presence of E. coli) zones of inhibition. The
reason may be that after 180 days the hydrogel samples
stored at 40
C became totally dry and, as a result, BA was
detached from the base of the hydrogel (as shown in
Figure 2), which caused no or uneven distribution of BA
in the dry hydrogel as well as the formation of an unclear=
uneven zone of inhibition.
Thus, it is clear that boric acid retains its activity in the
hydrogels for the whole period of investigation (180 days).
It is known that boric acid is the product of a third oxidation
of boranes, which is a very stable and relatively benign com-
pound to humans [32]. Both the solid and solution of boric
acid (in water) are very stable at room temperature and
stable for an indefinite period [33].
 156
4. Conclusions
The PVP-CMC and PVP-CMC-BA hydrogels are already
proven to be useful and effective from the health-care point
of view and they are very promising, user-friendly, and safe
to use as wound-dressing materials. The above-mentioned
experiments and their results give information about the
biological and environmental stability of the hydrogels.
Considering all the points, preserving the hydrogels at
5
C  2
C (in the refrigerator) seems to be a very appropri-
ate condition for storage. It can be observed from all the
results that the properties (moisture content, physical
appearance, sterility) of both PVP-CMC and PVP-CMC-
BA hydrogels at 5
C  2
C do not vary significantly during
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the entire period of study (180 days). Thus the temperature
range 5
C  2
C can be recommended as the best storage
condition for these two novel medicated hydrogel dressings
to maintain biological as well as environmental stability.
Acknowledgment
The authors are thankful to the Ministry of Education,
Youth and Sports of the Czech Republic (MSM
70088352101) for financial support. This article was also cre-
ated with the partial support of the Operational Programme
Research and Development for Innovation co-funded by the
European Regional Development Fund (ERDF) and the
national budget of the Czech Republic within the framework
of the Centre of Polymer Systems project (reg. number:
CZ.1.05=2.1.00=03.0111). Moreover, the authors are also
thankful to Dr. Vera Kasparkova for providing valuable
suggestions during the planning of experimental design for
stability study of medicated hydrogels.
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