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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 specified condition within which the product still meets its
established specifications [18]. Stability is an essential factor
The concept of hydrogels used in medical applications is not to maintain the quality, safety, and efficiency of a medical
new. Hydrogels were first introduced for clinical purposes in device. A product that is not of a sufficient stability can
ophthalmological use in the early 1950s by Wichterle and result in changes in physical (hardness, dissolution rate,
Lim [1]. Hydrogels have various applications in biomedicine phase separation, etc.) as well as chemical characteristics
[2–6]; among them, hydrogels for wound dressing have one (formation of high-risk decomposition substances). Micro-
of the major implementations in this domain. Hydrogel biological instability of a sterile product could also be
use for wound dressing was invented in the late 1980s by hazardous [18]. The stability study consists of a series of tests
Rosiak et al. [7,8]. Hydrogels can be successfully used as in order to obtain an assurance of stability of the product
dressing materials in medical treatment for burns and packed in its specified packaging material and stored in the
wounds because a wet environment can enhance the wound- established storage condition within the determined time
healing process [9–12]. Due to their high water content, period. Hydrogels were prepared in combination with syn-
hydrogels possess excellent biocompatibility and a degree thetic and natural polymers like polyvinylpyrrolidone
of flexibility similar to natural tissues, minimizing potential (PVP) and carboxymethyl cellulose (CMC), along with other
irritation to surrounding membranes and tissues [13–16]. ingredients, and boric acid (BA) was incorporated within the
Thus, the main goal of wound-dressing hydrogels is for hydrogel to acquire antimicrobial property within it.
application on the patients’ skin lesions [17]. It should be Further, it has been established that both PVP-CMC and
noted that medical device use has expiration dates, and use PVP-CMC-BA hydrogels are ideal for wound dressings
needs to take place before those dates. After that period, it and recommended for their safe use in humans=animals after
can be dangerous and may cause harm. The adverse effect thorough examination and characterization of such aspects
can be mild to severe. Therefore, when hydrogels are con- as swelling; moisture content; moisture-retention capacity;
sidered for use in medical devices, it is necessary to know ability for water vapor transmission; internal and external
their durability for safe use. The durability highly depends morphology; physico-chemical, mechanical, viscoelastic,
on the environmental conditions under which the material and antimicrobial properties; microbial penetrability and
was stored. It is very important to know the appropriate biocompatibility with respect to cytotoxicity; skin irritation
storage conditions which are suitable for the longer stability and skin sensitization tests, etc. [19–26]. Finally, it is impor-
of the materials. The objective of a stability study is to deter- tant to examine the storage condition of the hydrogels
mine the shelf-life, namely the time period of storage at a during product delivery. Moreover, it is essential to preserve
the valuable properties of hydrogels specifically prepared for
Address correspondence to: Nabanita Saha, Polymer Centre, health-care purpose. Thus, special attention needs to be
Centre of Polymer Systems, Tomas Bata University in Zlin, given to their biological and environmental stability. The
Nam T. G. Masaryka 5555, 76001 Zlin, Czech Republic. aim of this study is to identify the durability or stability of
E-mail: nabanitas@yahoo.com; nabanita@ft.utb.cz the hydrogels under different storage temperatures and to
 Stability Study of Hydrogel Wound Dressings 151

find out the appropriate storage condition for the novel the hydrogels was recorded after each specific time interval
hydrogel wound dressings. The present paper focuses on for the samples from each storage condition and then the
the occurrence of changes observed in PVP-CMC and samples were dried under room temperature until they
PVP-CMC-BA hydrogels at different storage temperatures assumed constant weight and then the measurements were
on the basis of their significant existing properties: physical done. In each case three replicates were taken and the
appearance (morphology, thickness, size, texture, color); average value of moisture content was determined.
moisture content; preservation of sterility; and retention of
ðMn Þ% ¼ ½ðWw  Wd Þ=Ww   100 ð1Þ
boric acid activity (i.e., on the basis of antibacterial effective-
ness), etc.
2.5 Physical Appearance
2. Experimental Changes in physical appearance of the material can arise due
to the deviation from the established specifications; i.e., it
2.1 Materials for Hydrogel Preparation refers to the fact that the material is not more stable and
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Polyvinylpyrrolidone K 30 (PVP: molecular weight 40,000), reached the end of its shelf-life. Three parameters were
polyethylene glycol 3,000 (PEG: average molecular weight checked periodically for 180 days: (a) Weight of the samples
3,015-3,685), and agar were obtained from Fluka, Switzer- was measured by analytical balance and data was recorded;
land; sodium carboxymethyl cellulose (CMC) was purchased (b) size and thickness of the hydrogels were measured using a
from Sinopharm Chem. Reagent Co., Ltd, China; boric acid ruler and micrometer; (c) color, texture, and appearance
(BA) from Sigma-Aldrich, USA; and glycerin obtained from were observed visually and any differences were noted.
Lachema Ltd, Czech Republic.
2.6 Preservation of Sterility
2.2 Preparation of Hydrogels Maintenance of the sterility is one of the most important
The ingredients used for the preparation of hydrogels were factors for medical devices. As long as the sterility of the bio-
PVP, CMC, BA, PEG, agar, and glycerin. The polymer solu- material persists, no microbe can grow on the hydrogel.
tions were prepared in sealed bottles by dissolving weighed Thus, to confirm the stability of the hydrogel from a biologi-
components in water. Then, moist heat treatment [15 lbs cal point of view, the hydrogels were checked time to time
(107 kPa) pressure, 120
C, 20 minutes] [19,20] was applied with careful visual observation, i.e., appearance of any
to the solutions, followed by solution (5 ml each) casting in microbial colonies on the hydrogel.
molds of 50 mm diameter. The polymer solutions were
allowed to cool at room temperature (22
C  3
C) under 2.7 Retention of Boric Acid Activity in PVP-CMC-BA
an aseptic environment to achieve the desired hydrogels. Hydrogel
The hydrogel without BA was designated ‘‘PVP-CMC’’
BA was incorporated in the hydrogel to achieve medicinal
and the hydrogels with BA as ‘‘PVP-CMC-BA.’’ The initial
value and antimicrobial property within the hydrogel. The
composition of each test samples is given in Table 1.
dose of BA in the hydrogel was maintained at 3% as it is
the permissible limit of BA for health-care purposes
2.3 Experimental Parameter/Testing Methods [20,28]. It is essential to check the persistence of BA in stable
To identify the suitable storage conditions for PVP-CMC form as BA is the responsible ingredient to preserve the anti-
and PVP-CMC-BA hydrogels, some selected tests such as microbial property of hydrogel. If BA degrades with time, it
retention of moisture content, changes in physical appear- will lose its antimicrobial activity and destabilize the hydro-
ance=behavior, preservation of sterility, retention of BA gel. The retention of BA activity was assayed on the basis of
activity, etc., were carried out and are mentioned below. FTIR measurement as well as on the basis of antibacterial
effectiveness (measuring the zone of inhibition).
2.4 Measurement of Moisture Content [19,27] 2.7.1 FTIR of PVP-CMC-BA Hydrogel
PVP-CMC-BA hydrogels in dry form were analyzed by
Moisture content of the hydrogels was measured gravimetri- FTIR. The ATR-FTIR spectroscopic analysis was conduc-
cally using Equation (1) in which Mn ¼ moisture content (%) ted using a NICOLET 320 FTIR Spectrophotometer with
of material n, WW ¼ wet weight of the sample, and ‘‘Omnic’’ software package over the range 4000–600 cm1
Wd ¼ weight of the sample after drying. The wet weight of at room temperature. A uniform resolution of 2 cm1 was
maintained in all cases. The FTIR spectra values between
Table 1. Composition of hydrogels (W=V %) [20 and 23] 1800–1000 cm1 have been considered to evaluate the
stability of BA.
Sample
Index PVP CMC PEG Agar Glycerin BA Water 2.7.2 Antimicrobial Assay of PVP-CMC-BA Hydrogel
The antimicrobial properties of PVP-CMC-BA hydrogels
PVP-CMC 0.2 0.8 1 2 1 0 95 were investigated by agar diffusion method [20,29–31]. Anti-
PVP-CMC- 0.2 0.8 1 2 1 3 92 microbial characteristics of these hydrogels were examined
BA on the basis of the dimension of the inhibition zone
 152 N. Roy et al.

generated in the presence of pathogenic bacteria like Escher- 3.2 Physical Appearance of Hydrogels
ichia coli and Staphylococcus aureus. The antibacterial assay
Physical appearance of the materials offers lots of infor-
was conducted in a sterile nutrient agar (3%) medium, which
mation concerning the stability of hydrogels. Figure 2 shows
was inoculated with the above-mentioned bacterial strains
the optical view of the initial samples and the samples after
under aseptic condition. After solidification of nutrient agar,
180 days of storage at different temperatures for PVP-CMC
a piece of hydrogel (15 mm diameter) obtained from three
and PVP-CMC-BA hydrogels. Initial samples of PVP-CMC,
different storage conditions (5
C  2
C, 22
C  3
C, and
when dried at room temperature, appear transparent, but in
40
C) was placed on the surface of the medium. The testing
the case of dry PVP-CMC-BA white dots of BA appear
plates were then incubated in a temperature-controlled incu-
throughout the film [20]. From Figure 2, it is noticeable that
bator at 37
C to observe the effect of PVP-CMC-BA hydro-
after 180 days at 40
C the PVP-CMC-BA hydrogel becomes
gels on the growth of individual bacteria. The antimicrobial
totally dry and brittle, and BA comes out because of the effect
assay was carried out with the PVP-CMC-BA hydrogel sam-
of the high temperature. The drying process of the hydrogels
ple isolated from different temperatures (5
C  2
C, 22
C 
become faster at higher temperature, and during drying water
3
C, and 40
C) at different intervals (i.e., 0, 45, 90, 180
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evaporates from the hydrogels and, after a certain time, the

days). The persistence of antibacterial activity of BA was
dissolved BA reaches its saturation and re-crystallizes, which
evaluated on the basis of zone of inhibition.
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
3. Results and Discussions PVP-CMC-BA, at 40
C, from 90 days onwards white spots
3.1 Moisture Content of Hydrogels of BA appeared and it were spread like a network throughout
the hydrogels. This appeared because, at higher temperature,
Moisture-holding capacity is one of the most important the hydrogels started to lose water and oversaturated BA
properties for hydrogels. Because of it, hydrogels are able became visible. In the case of both hydrogels, the rate of
to provide a moist environment to the wound; moreover, it weight loss (Figure 3) and reduction of thickness (Figure 4)
is proven that the moist environment helps to heal the are remarkably higher for the samples stored at 40
C; conse-
wound faster [9–12]. Thus, evaluation of the moisture- quently the same trend has been observed in the reduction of
retention capacity of both PVP-CMC and PVP-CMC-BA the diameters of the hydrogels. The changes are moderate for
under different storage conditions is important and is shown the samples at 22
C  3
C, but very little at 5
C  2
C
in Figure 1. It can be seen from Figure 1 that under because of the variation in temperature. The rate of evapor-
5
C  2
C (in refrigerator) both the hydrogels lose very ation of water from the hydrogels varies in the following man-
nominal amounts of water up to 180 days of study. Even ner: 5
C  2
C < 22
C  3
C < 40
C.
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. 3.3 Preservation of Sterility of Hydrogels
After 180 days, all the PVP-CMC-BA samples were found to
be totally dry. At higher temperature (40
C), the water from The appearance of microbial colonies on the material refers
the hydrogels evaporates faster, which causes higher degree to the fact that the material is no longer sterile, which means
of moisture loss from the hydrogels, thus becoming almost it loses its biological stability. Thus, it is important to check
or totally dry after 180 days. 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-
Fig. 1. Effect of storage conditions on moisture content of microbial agent and it resisted the hydrogels from any
hydrogels. (Figure is provided in color online.) 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

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
Fig. 4. Effect of storage conditions on the thickness of hydro- peak of PVP and 1,588 cm1 peak of CMC become a single
gels: (a) PVP-CMC; (b) PVP-CMC-BA. (Figure is provided in broad peak at 1632 cm1 [20]. The intensities of the peaks at
color online.) 1632 cm1 of the PVP-CMC-BA hydrogel samples vary little
among themselves. Also, the 1280 cm1 peak of C-N stretching
3.4 Retention of Boric Acid Activity in PVP-CMC-BA vibration of PVP and 1062 cm1 peak of 1,4-b-D-glucosidic
Hydrogel stretching vibration of CMC [19,20] in initial PVP-CMC-BA
3.4.1 FTIR of PVP-CMC-BA Hydrogel either diminish or shift for the samples after 180 days.
Therefore, it can be mentioned that the storage temperatures
The FTIR spectra of PVP-CMC-BA hydrogels (initial and the
do not affect the activity of BA throughout the experimental
samples after 180 days of storage) were studied in a dry state to

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 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)
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or unclear (in the presence of E. coli) zones of inhibition. The

Fig. 6. FTIR spectra of PVP-CMC-BA hydrogels (in dry form) reason may be that after 180 days the hydrogel samples
stored under different storage conditions. (Figure is provided in stored at 40
C became totally dry and, as a result, BA was
color online.) 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=
period, but it may affect up to a certain extent the properties of uneven zone of inhibition.
base polymers like PVP and CMC, as they are biomaterials. Thus, it is clear that boric acid retains its activity in the
hydrogels for the whole period of investigation (180 days).
3.4.2 Antimicrobial Assay of PVP-CMC-BA Hydrogel
It is known that boric acid is the product of a third oxidation
Boric acid (BA) was incorporated into the PVP-CMC hydro- of boranes, which is a very stable and relatively benign com-
gel to achieve an antimicrobial-property-based hydrogel. pound to humans [32]. Both the solid and solution of boric
The PVP-CMC-BA hydrogel containing 3% BA shows very acid (in water) are very stable at room temperature and
good resistant property against microbes [20]. Thus, it is stable for an indefinite period [33].
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.)
 156 N. Roy et al.

4. Conclusions 10. Cartmell, J. V.; Wolf, M. J.; Allaire, M. J.; Sturtement, R.

Hydrogel Burn Dressing Product; U.S. Patent, US005115801A,
The PVP-CMC and PVP-CMC-BA hydrogels are already 1992.
proven to be useful and effective from the health-care point 11. Martineau, L.; Shek, P. N. Burns. 2006, 32, 172.
of view and they are very promising, user-friendly, and safe 12. Pal, K.; Banthia, A. K.; Majumdar, D. K. J. Mater. Sci: Mater.
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The authors are thankful to the Ministry of Education, Patent 302405, 2011.
Youth and Sports of the Czech Republic (MSM 23. Roy, N.; Saha, N.; Kitano, T.; Lehocky, M.; Vitkova, E.; Saha, P.
70088352101) for financial support. This article was also cre- Int. J. Polym. Mater. 2012, 61, 72.
ated with the partial support of the Operational Programme 24. Roy, N.; Saha, N.; Humpolicek, P.; Saha, P. Soft Mater. 2010,
Research and Development for Innovation co-funded by the 8(4), 338.
25. Molina, M. J.; Pierola, I. F.; Gomez-Anton, M. R. Int. J. Polym.
European Regional Development Fund (ERDF) and the
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