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.