4 RESULTS AND DISCUSSION
Recently, research attention has drawn towards polymer nanocomposite due
to its various applications and capabilities like biocompatibility,
degradability, and non-toxicity (Ma et al., 2017). Nanocomposites are
defined typically as novel materials where at least one of the
constituents is in between the number range of 1 to less than 100 nm.
Due to the nanocomposites’ small size effect, the physical and chemical
properties are better than the traditional composites. With the
advancement of nanoscience and nanotechnology, remarkable innovations
towards nanocomposites have been accomplished (Huang & Cheng, 2017).
Nanocomposites are also known as one of the most promising materials in
the 21st century for its rapid development and is vastly utilized in
many disciplines because of its unique polymer attributes such as
lightweight and helps ease production along with processing (Ma et al.,
2017).
In this study, PEG-chitosan nanocomposite was selected as a wound
dressing and drug delivery medium. The developed hydrogel showed a
uniform light blue color. The hydrogel was transparent. PEG-chitosan
nanocomposites are widely recognized as biodegradable polymers with
outstanding safety and biocompatibility. They are used in association
with other polymers to produce controlled-release drugs. The second
prolific polysaccharide in nature is chitosan, the cationic
(1-4)-2-amino-2-deoxy-β-D-glucan. Chitosan holds great significance due
to its biodegradability, non-toxicity, and biocompatibility (Gilani et
al., 2018). Surface coating by means of biodegradable and biocompatible
polymers with low toxicity like chitosan and PEG were applied to curb
the phagocytic reactions and improve the lifespan of the nanoparticles.
Intriguingly, chemical alteration of chitosan and PEG does not only
enhance the biocompatibility of chitosan, but also decreases the
adsorption of circulating plasma proteins upon the material surface
(Rabou & Ahmed, 2017).
Microscopic examination is important to observe the surface morphology
of the developed hydrogels. Figure 1 represents the SEM micrographs of
the nanocomposite hydrogel with phomopsidione. The hydrogel showed a
smooth and uniform surface. Phomopsidione was successfully embedded in
the smooth-textured surface of the hydrogel. Figure 2 depicts the TEM
micrographs of the hydrogel produced. On the micrograph, nanospheres of
various sizes were observed, with no sign of aggregation. Nanospheres
ranging from 16.9 ± 3.8 nm represented chitosan whereas nanospheres
ranging from 4.8 ± 1.6 nm represented PEG, based on the comparison with
the previous studies (Liu & Kim, 2012). The mixing of chitosan and PEG
was homogenous. All nanospheres observed were less than 100 nm in
diameter. Supporting these results, Huang and Cheng reported that
nanocomposites are component ranges from 1 to less than 100 nm (Huang &
Cheng, 2017).
The FTIR spectra of chitosan, PEG, hydrogel with phomopsidione and
negative control hydrogel were studied (Figure 3). The spectrum of
chitosan showed characteristic absorptions at 3283 (overlap of O-H and
N-H stretching), 1646 (C-N stretching) and 1558 (N-H bending)
cm-1 (Manuela et al., 2016). For PEG, absorption bands
at 1359 and 2879 cm-1 were due to the bending and
stretching vibrations of C-H (Shameli et al., 2012). In the spectrum of
negative control, the molecular interactions between the chitosan and
genipin-crosslinked PEG caused the shifting in the absorption bands. The
characteristic absorptions of chitosan were shifted to 1636 and 1556
cm-1 while the absorption at 3283
cm-1 became stronger at 3278
cm-1.With the presence of phomopsidione in the
nanocomposite, the shift in the peaks towards lower frequency at 1600
and 1554 cm-1compared to the negative control. The
results suggest the successful binding of phomopsidioneto the
nanocomposite hydrogel.
This drug release study is important to determine the drug release
behavior of phomopsidione form the hydrogel, by quantifying the
phomopsidione released with HPLC. The test medium was artificial sweat
solution prepared at pH 5.5 as to mimic the condition of the skin
(Manuela et al., 2016). Figure 4 shows the pattern of phomopsidione
released from the hydrogel for a period of 72 hours. Overall, no burst
release was observed throughout the whole experimental period. The
release of phomopsidione was slow and gradual, with an average amount of
34 μg/mL phomopsidione released per hour. The release followed the first
order of kinetic, where 67.9 ± 6.4% of phomopsidione was released. This
is due to excellent drug carrier properties from the two polymers,
chitosan, and PEG. Nano-sized chitosan showed great promise in drug
delivery as they possess high surface to volume ratio and porosity which
enhances its drug loading capacity and deliver applications. Moreover,
Parveen and Sahoo mentioned that surface coating by polymers such as
chitosan and PEG were used to enhance the lifespan of the nanoparticles
(Parveen & Sahoo, 2011). Chitosan accepts protons at low pH upon adding
cationic polymers and becomes positively charged (Gupta, Vermani, &
Garg, 2002; Saikia, Gogoi, & Maji, 2015). This type of polymer swells
at low pH mediums. Since an acidic (pH 5.5) medium was used in this
experiment, it caused swelling of the polymeric matrix which in turn
released the loaded drug into it. Genipin was added in the nanocomposite
as a crosslinker between phomopsidione and polymers by forming physical
bonding such as van der Waal forces, hydrogen bonding and ionic
interaction (Manickam, Sreedharan, & Elumalai, 2014). It plays a
significant role in controlling the drug release from the hydrogel. The
drug release behavior study showed that genipin is an excellent
crosslinker for phomopsidione.
The mechanical properties of the hydrogels were determined to evaluate
their suitability as a wound dressing material for chronic wounds. The
average tensile strength for hydrogel with phomopsidione was 15.46 MPa
which showed an increment of 29.62% in average tensile strength as
compared to that of the negative control (Table 1). In addition, the
results showed a 5.41% increment in elongation for the hydrogel with
phomopsidione. Lastly, Young’s modulus for hydrogel with phomopsidione
was 1152.69 MPa which showed an increment of 13.76% from the negative
control. The developed nanocomposite hydrogel was soft, flexible and
elastic, which is ideal as a wound dressing material (Tong et al.,
2017). The elasticity of the nanocomposite hydrogel is crucial to hold
the wound dressing in place for a long period of time, in order to
provide an excellent therapeutic effect for the wound recovery (Ali &
Ahmed, 2018). The loading of phomopsidione into the hydrogel did not
cause a significant change in the elongation at break and Young’s
modulus of the hydrogel (p ≥0.05). However, the tensile strength
of the hydrogel was significantly improved with the addition of
phomopsidione (p ≤0.05). The improved tensile strength of the
nanocomposite hydrogel means that it is able to endure greater force
before it breaks. Pressure ulcers are constantly exposed to pressure or
external forces hence a wound dressing with better tensile strength is
favorable.
By comparing to Liu and Kim, the Chitosan-PEG blend hydrogels were found
to be more flexible and possess better mechanical properties, as
compared to pure chitosan films (Liu & Kim, 2012). A study by Suyatmaet al . reported that a pure chitosan film exhibited a tensile
strength of 82.4 MPa with 5.2% elongation at break whereas the study by
Liu and Kim showed that the Chitosan-PEG exhibited a tensile strength of
85.5 MPa with elongation at break of 25.5%, which justified enhanced
properties upon mixing (Suyatma et al., 2004; Liu & Kim, 2012). This is
because when the two polymers are blended together, the chitosan helped
in improving mechanical properties while PEG improves its flexibility
(Huang & Cheng, 2017). In comparison to other blends, Zivanovic,
Davidson & Kit reported that chitosan-polyethylene oxide blend of the
same mixing ratio exhibited a mean tensile strength of 55.4 MPa with an
elongation at break of 10.8% (Zivanovic, Davidson & Kit, 2007).
Ultimately, the addition of phomopsidione has shown improvement in the
nanocomposite hydrogels’ mechanical properties. Therefore, the
nanocomposite hydrogels, comprised of chitosan and PEG blends, have
shown great potential to serve as antimicrobial wound dressings. The
mechanical properties of the nanocomposite hydrogels can be further
improved. Hydrogel layering can help improve the overall mechanical
properties. Also, manipulating the thickness of the nanocomposite
hydrogels can ultimately find the optimum mixing ratio giving the best
mechanical properties (Zivanovic, Davidson & Kit, 2007).
Various natural polymers were utilized as wound dressing material.
However, the application of chronic wounds is limited due to poor
swelling activity. Overall, the maximal swelling ratio of the
nanocomposite was high, which make it an ideal wound dressing material.
The high swelling ratio of the hydrogel allows the absorption of a large
amount of exudates in chronic wounds (Qin, 2008). The high swelling
ratio is comparable to carboxymethyl cellulose hydrogel (Chang et al.,
2010). The excellent swelling ratios are contributed by many hydrophilic
groups in the hydrogel, particularly the ionic bond between water
molecule and the amino groups of chitosan (Kiuchi, Kai, & Inoue, 2007).
The difference in swelling ratios for nanocomposite hydrogel with
phomopsidione and negative control were not statistically significant
(p ≤0.05). The addition of phomopsidione into the hydrogel did not
affect its swelling ratio.
The antimicrobial study was performed to evaluate the antimicrobial
efficiency of the developed hydrogel on wound pathogens. The study was
done through the parallel streak method with 20 mm by 20 mm hydrogels.
The inhibitory activity on all test microorganisms is shown in Table
2. The hydrogel with phomopsidione showed significant
antimicrobial activity against all Gram-negative bacteria and one yeast
(C. utilis ). The antimicrobial activity of the hydrogel was a
narrow spectrum. The largest inhibition zone observed was againstP. aeruginosa with an average inhibition zone diameter of 41.2
mm. P. aeruginosa is most frequently isolated from chronic wounds
treated with antibiotic therapy (Serra et al. 2015). Due to its high
virulence, the bacterium often prolongs the hospitalization of patients
with the chronic wounds. Negative control hydrogels were also tested on
and were done as comparative studies. It was observed that the hydrogels
without phomopsidione showed no antimicrobial effects. This has proven
that the synthesized hydrogels were able to release efficiently onto the
agar medium as well as to suppress any microbial growth.
Besides, opposing with the previous reports by Fayaz et al . and
Bhawana et al . the hydrogel with phomopsidione exhibited
inhibitory activity only on Gram-negative bacteria (Fayaz et al., 2010;
Bhawana et al., 2011). However, the inhibitory activity of phomopsidione
on Gram-negative bacteria was previously reported earlier (Sahadan et
al., 2019). Phomosidione did not target the bacterial cell wall. Figure
5 depicts the antimicrobial effect of the nanocomposite hydrogels
against C. utilis . Similarly, the presence of clear zones from
phomopsidione against Candida species also reported (Tong et al.,
2017).
The quantitative evaluation of the antimicrobial efficiency was
performed with the Hohenstein challenge test. Four test microorganisms
showed 99.9% of bacterial reduction, relative to the negative control.
The results were tabulated in Table 3. Based on the results, the
hydrogel with phomopsidione has proven to exhibit a significant
reduction in the growth of Gram-negative bacteria and yeast. The outcome
of this study is in agreement with Tong et al . who reported
phomopsidione’s biocidal effect towards yeast, though a similar effect
was observed form Gram-negative bacteria (Tong et al., 2017). To support
the claim, the hydroxyl groups which are present in a bioactive compound
served to reduce free radicals from the microorganisms by donating their
hydrogen atoms and this inhibits microbial growth (Feng & Liu, 2009).
Since phomopsidione contains hydroxyl groups, free radicals from the
microorganisms were able to be scavenged resulting in the inhibition of
microorganisms. Mirroring the results of parallel streak test, the
highest percentage of inhibition was observed on P. aeruginosa .
The results showed the potential use of this hydrogel in the treatment
of Gram-negative infection on chronic wounds.