Cs-g-PVP was obtained by grafting 4-vinylpyridine copolymerization onto chitosan as described in the experimental section. According to the given equations in the experimental section, the grafting (G %) and grafting efficiencies (GE%) were calculated to be 92 and 236 %, respectively. FTIR spectroscopic analysis was used to confirm the grafted copolymer that had been formed. In addition, Cs-g-PVP/f-MWCNTs hybrids containing 3wt% and 5wt% of f-MWCNTs were also prepared. The affinity of the obtained grafted copolymer and its hybrids for copper ions adsorption was determined using batch adsorption method and metal removal % was determined using atomic absorption spectrometry (AAS). The effect of pH and the adsorbent mass on % metal removal was studied. Thermal stability of the copolymer, Cs-g-PVP and the two hybrids was determined via thermal gravimetric analysis, TGA, whereas scanning electron microscopy was applied for morphological studies of these samples. Antimicrobial activity of the all-prepared samples was evaluated.
3.1. FTIR analysis
The evidence of the formation of Cs-g-PVP was achieved by FTIR analysis. Figure 1 (a,b) represented the FTIR spectrum of both Cs and Cs-g-PVP. It was clearly shown a strong IR band at 3433 cm-1 which should be assigned to the symmetrical stretching vibration of the OH groups, the extension vibration of the NH2, and the intermolecular hydrogen bonds of the polysaccharide. There are also two IR peaks appeared at 2918 and 2851 cm-1 that may be due to the aliphatic CH2 and CH. The C-O absorption peak of the hydroxyl group was observed to 1068 cm-1. In Figure 1, the grafting of 4-vinylpyridine onto chitosan was confirmed by the presence of C=N groups band at about 2120 cm−1 from the poly 4-vinylpyridine chains and the –CH bending of the aromatic ring of the vinyl pyridine that appeared around 746, 821, and 1601 cm-1 and this is very indicative to the grafting copolymerization of vinyl pyridine moiety with chitosan [34].
3.2. Thermogravimetric analysis
TGA analysis of Cs, Cs-g-PVP, Cs/f-MWCNTs and Cs-g-PVP/f-MWCNTs hybrid that containing 5% by weight of f-MWCNTs are shown in Figure 2. The thermogram revealed that Cs-g-PVP has a relative higher thermal stability than Cs in terms of both initial decomposition temperature (IDT) and residual weight below 500 oC,. The effect of f-MWCNTs (5wt% by weight) in both Cs and the grafted copolymer was investigated, and the results revealed that both Cs/f-MWCNTs and Cs-g-PVP/f-MWCNTs hybrids had significant higher thermal stability than either Cs or Cs-g-PVP. For example, at 250 ᵒC, the recorded weight loss of Cs, Cs-g-PVP, Cs/MWCNTs and Cs-g-PVP/MWCNTs hybrids are 27%, 31%, 13% and 10% respectively. The improved thermal stability of the two hybrids was found to be so significant at the decomposition temperature of 300 oC. The weight loss of Cs/f-MWCNTs and Cs-g-PVP/f-MWCNTs hybrids are 21% and 11% whereas the percentages weight loss of both Cs and Cs-g-PVP are 57 and 49 respectively. Cs and Cs-g-PVP hybrids lost 66% and 57% of their weight respectively, at 400 oC while Cs/f-MWCNTs and Cs-g-PVP/f-MWCNT hybrids lost 47% and 43% of their weight at the same temperature. At 515 oC, both Cs/f-MWCNTs and Cs-g-PVP/f-MWCNTs hybrids have almost the same weight loss of about 58 %, whereas Cs and Cs-g-PVP exhibited weight loss of 88 and 75 %, respectively. The thermal properties of the samples under investigation are shown in Table1. It showed the residual weights of the tested samples at 400 and 500 ᵒC, as well as the temperatures at which they lost 10 and 50% of their weight. The observed weight loss in the early stages of degradation for all investigated samples might be attributed to any attached water residues or volatiles being released from the polymeric chains. The observed degradation of Cs could be due to dehydration and depolymerization of Cs units, which increases as the polymeric network structure proceeds [35-36]. The presence of a PVP block in the grafted copolymer, with its better thermal stability due to the presence of the aromatic ring in the chemical structure of PVP, could explain the higher thermal stability of Cs-g-PVP than Cs. It was demonstrated that the thermal stability of Cs and the grafted copolymeric matrix of Cs-g-PVP was improved by incorporation f-MWCNTs in 3wt% and 5wt% by weight, resulting in two hybrids. The barrier function of the f-MWCNTs may be responsible for this improved thermal stability.
Table 1. Thermal properties of various investigated samples
Sample
|
T10%
|
T50%
|
R400%
|
R500%
|
Cs
|
53
|
277
|
34
|
13
|
Cs-g-PVP
|
207
|
310
|
43
|
26
|
Cs/MWCNTs (3wt%)
|
213
|
423
|
53
|
43
|
Cs-g-PVP/ MWCNTs (5wt%)
|
229
|
436
|
57
|
43
|
3.3. Scanning electron microscopy
SEM images of Cs and Cs-g-PVP Cs-g-PVP/f-MWCNTs (I and II) hybrids and are shown in Figure 3 (a-d). The morphology of the chitosan was depicted in Figure 3 (a). Upon graphitization of Cs with 4-VP, to obtain Cs-g-PVP, an obvious change in the surface morphology of the grafted chitosan that gave a proof for conjugation of 4-VP blocks in the copolymer chains and this is represented in Figure 3 (b). By treating chitosan grafted- 4-vinylpyridine copolymer with f-MWCNTs (3wt% and 5wt%,). It was apparently shown that the functionalized MWCNTs are bonded to chitosan-g-PVP. The f-MWCNTs was appeared in micron length sizes with distinct agglomeration in the surface of the formed polymeric composite.
3.4 Batch adsorption studies
Batch adsorption experiments were performed using Cs-g-PVP, Cs-g-PVP/f-MWCNTs hybrid (containing 3wt% and 5wt% by weight) as polymeric adsorbent according to the method described before in the experimental part. Cs and Cs/f-MWCNTs was also examined as metal adsorbent for comparison. Metal solutions with concentration of 5x10-3 mol/L of CuCl2. 2H2O at pH (4, 7 and 9), adsorbent copolymer (0.05, 0.10, 0.15 and 0.20 g) were applied and the effect of both pH and copolymer mass will be discussed.
3.4.1. Effect of pH and adsorbent mass on metal removal by the graft copolymers Cs-g-PVP
The results of copper (II) uptake by the produced Cs-g-PVP as a function of pH and mass adsorbent are shown in Figure 4. The applied masses of the grafted copolymer as adsorbent are; 0.05, 0.10, 0.15 and 0.20 g whereas the working pH values are 4, 7 and 9. The results show that when different masses of adsorbent were used, the metal removal % increase as the pH increase from 4 to 7 [37], but there was a considerable reduction at pH 9. Figure 4 indicates that both the pH and the adsorbent mass have an impact on the metal removal % of copper (II) from aqueous solutions. For adsorbent masses of 0.05, 0.10, 0.15, and 0.20 g, the metal removal % was 21.08, 26.22, 28.75, and 29.17 at pH 4. At pH 7, the percentages of metal removal for the same copolymer masses are 35.54, 38.38, 39.32, and 40.09. It is noticed that at pH 9 these percentages are reduced to be 30.27, 32.17, 34.59 and 35.12. The increase of metal removal by the increase of pH from 4 to 7 may be due to the remarkable hydrolysis of the copper (II) ions at higher pH and this may compete with polymer chelation process. In all cases, the pH highly affects the oxygen atoms of OH or the lone pairs of electrons on the nitrogen atoms of the adsorbent. At higher values of pH, the OH may lose its proton and bear a negative charge while the nitrogen atom lone pair will be more available for binding to metal ions and this increase the attraction of metallic ions onto the surface of adsorbent [38]. This decrease occurred at pH 9 for all masses of the adsorbent which may be due to the precipitation of the metal hydroxides in the basic solution [39]. The given results show also that at all pH values, the % of metal removal is increased with the increase of the copolymer mass. At pH 7, the metal removal percentages are 35.54%, 38.38%, 39.32% and 40.09% for the adsorbent masses of 0.05 g, 0.10 g, 0.15 g and 0.2 g respectively. It noticed also that there is slight increase in the metal ion uptake on increasing the copolymer mass from 0.15 g to 0.20 g. The increase of the removal of Cu(II) ions with increasing the mass of adsorbent which is directly proportional to the adsorption capacity by the grafted copolymer, may be due to the increase of the polymer sites available for chelation. The effect of the mass of adsorbent on the extent of metal removal was also investigated and the results reveal that removal of Cu (II) ions increases with increasing the mass of adsorbent. Results also show that the amount of metal ions removed by the graft copolymer, Cs-g-PVP, slightly increases with the increase of the mass of adsorbent from 0.15 g to 0.2 g. The increase of the metal removal, which is directly proportional to the adsorption capacity by the grafted copolymer, with increase of the mass of adsorbent may be due to the increase of the polymer sites available for chelation.
3.4 Metal removal by Cs-g-PVP/f-MWCNTs hybrids
Further investigations were performed for copper (II) removal using Cs-g-PVP/f-MWCNTs (containing 3wt% and/or 5wt% by weight) hybrids as metal adsorbent. Removal efficiency (R%) of metal removal by both Cs and Cs/f-MWCNTs are also given for comparison. Adsorbent mass fixed at 0.15 g at pH 7. The results are represented in Figure 5. The results show the % metal removal by the adsorbent, Cs-g-PVP/f-MWCNTs that containing 3wt% of the f-MWCNTs is 98.80 while the hybrid of 5wt% f-MWCNTs has metal removal % of 99.01. The ability of Cs and Cs/f-MWCNTs, containing 3wt% and 5wt% by weight, of f-MWCNTs was also investigated. The results indicated that the removal of Cu (II) ions from the aqueous solution reached 76.38, 84.36 and 89.19 for the three samples respectively. The observed high significant enhancement of metal removal % of these samples confirms the removal efficiency of f-MWCNTs which increases with increasing of its mass. This may be due to the presence of more negatively charged oxygen-containing groups on the oxidized or functionalized form of MWCNTs that have the affinity to attract the copper cations from the aqueous solutions [27].
The incorporation of Cu(II) in Cs-g-PVP and Cs-g-PVP/f-MWCNTs 5wt% was confirmed by SEM analysis, Figure 6 (a,c). Figure 6 (a) showed the presence of copper metal ions on the Cs-g-PVP polymeric surface that appeared as bright spots. Figure 6 (c) shows the SEM image of the as-prepared Cs-g-PVP/f-MWCNTs 5wt% after Cu(II) ions adsorption process. In figure 6 (c) shows that f-MWCNTs appeared as sequestered within regions of chitosan-g-PVP that probably considered as the more hydrophobic phase on the polymeric hybrid. The EDX profile showed peaks for C, N, O and Cu as elemental constituents of the grafted copolymer, Cs-g-PVP and Cs-g-PVP/f-MWCNTs after adsorption of Cu(II) (Figure 6b,d). In EDX, the chemical compositions of Cs-g-PVP/Cu in weight% showed (C, N, O and Cu) was found (57.2 wt%, 5.4 wt%, 25.4 w% and 12.0 wt%) (Figure 6c). However, the chemical compositions of Cs-g-PVP/f-MWCNTs/Cu showed (C, N, O and Cu) was estimated to be (56.1 wt%, 5.2 wt%, 26.5 w% and 12.1 wt%) (Figure 6d). Also mapping of each element in Cs-g-PVP/f-MWCNTs/Cu sample was represented in figure 6e. Thus, these results are confirming that the as prepared Cs-g-PVP and Cs-g-PVP/f-MWCNTs was an efficient material for Cu-adsorption in aqueous solutions.
Antibacterial activity
The antimicrobial activity of chitosan (Cs), chitosan grafted copolymer (Cs-g-PVP) and two grafted copolymers/f-MWCNTs hybrids (Cs-g-PVP/f-MWCNTs hybrids that containing 3wt% and 5wt% by weight of the MWCNTs) were evaluated against the gram +ve bacteria (B. Subtitles, St. aurous and St. faecalis) and the gram -ve bacterial strains (E. coli, N. gonorrhoeae and P. aeruginosa) that isolated from animal origin. Agar disk diffusion method was used for the determination of the preliminary antibacterial and Ampicillin was used as reference antibacterial drugs. All of the investigated samples showed, in vitro, antibacterial activity against the tested microorganisms. The results of antibacterial activity of the samples under investigation using inhibition zone method are listed in Tables 2.
The results showed that when compared to pristine chitosan, Cs-g-PVP had improved antibacterial activity against all strains of gram-positive and gram-negative bacteria. As a result, Cs has antibacterial efficacy against both types of bacterial strains in terms of inhibition zone diameter. The inhibition zone caused by Cs against the gram +ve bacteria (B. Subtitles, St. aurous and St. faecalis) are 13, 15 and 12 with efficiencies of 65, 83 and 67%, respectively with respect to the reference drug. On the other hand, the antibacterial activity of by the effect of Cs against the gram -ve bacterial strains (E. coli, N. gonorrhoeae and P. aeruginosa) are 14, 11 and 12 with efficiencies of 64, 55 and 71%, respectively when compared to the standard reference antibacterial drug. The insertion of 4-vinylpyridine (4-VP) as blocks via grafting copolymerization with Cs yielding Cs-g-PVP improved the antibacterial activity of this grafted copolymer against all tested microbes, according to the results of Table 2.
In comparison to the reference drug, the antibacterial efficiency for gram +ve bacteria (B. Subtitles, St. aurous, and St. faecalis) were 85, 94, and 83, respectively. The antibacterial activity of Cs-g-PVP against the gram -ve bacterial strains (E. coli, N. gonorrhoeae and P. aeruginosa) reached 86, 85 and 94%, respectively with respect to the reference antibacterial drug. As a result, the Cs-g-PVP exhibit better efficiency against G -ve bacterial strains than the G +v kinds, according to the given results. These results could be explained by the electrostatic interaction between positively charged chitosan molecules and negatively charged microbial cell membranes, which is the first proposed accepted mechanism for chitosan antibacterial activity [40].
The second predicted action mechanism is chitosan's binding to microbe DNA, which results in mRNA and protein synthesis suppression in the nuclei of bacteria due to chitosan penetration. [41]. Based on the well-known biological properties of nitrogen containing six membered aromatic heterocyclic compounds and their derivatives [42], the observed increased antibacterial activity of Cs-g-PVP can be attributed to the PVP blocks in the grafted copolymeric chains. Additionally, the hydrophobic character of 4-VP in the copolymer chains increases the density of positive charges on the system, which may favor microbial cell attachment to the antibacterial system. As a result, 4-VP-based polymers or copolymers are intriguing materials with a variety of uses, including antibacterial materials [43-44]. Despite its well-known antibacterial properties, applications of Cs are limited due to undesirable properties such as low solubility and chemical stability [45-47]. As a result, various modifications or the addition of reinforcement materials may give rise to improvement to the physicochemical properties of chitosan, allowing it to be used in a wider range of applications.
Multiwalled carbon nanotubes (MWCNTs) have thus been proposed as an excellent material to utilize as inorganic fillers for reinforcing or toughening polymeric materials [48], in addition to the grafting of 4-VP onto chitosan performance. Two samples of Cs-g-PVP containing 3 and 5% by weight of MWCNTs were functionalized as stated in the experimental section, affording two hybrids of Cs-g-PVP/f-MWCNTs for testing their biological activity. Table 2 shows the antibacterial activity of the two hybrids against the bacterial strains that were examined. When compared to either chitosan or the reference drug, the results showed that the two hybrids had much higher antibacterial efficacy against both types of bacteria, G +ve and G -ve bacteria. Antibacterial activity of the hybrid containing 3% by weight of f-MWCNTs against G +ve bacterial strains (B. Subtitles, St. aureus, and St. faecalis) was 95, 106, and 94 percent, respectively, as compared to the conventional drug.
In comparison to the reference drug, the obtained antibacterial activity of the same sample against G -ve bacteria (E. coli, N. gonorrhoeae, and P. aeruginosa) was 114, 110, and 106 respectively. The antibacterial activity effectiveness of the other hybrid, which contained 5wt% by weight of f-MWCNTs, was the highest of the investigated samples. In comparison to the reference antibacterial drug, the inhibitory effect of this hybrid revealed inhibition zones of 20, 21, and 17 mm, respectively, with efficiency of 100, 117, and 94 % against G +ve bacteria (B. Subtitles, St. aurous, and St. faecalis). When compared to the reference drug, the examined hybrid recorded inhibitory zones of 26, 24 and 21 mm for the G -ve bacterial strains (E. coli, N. gonorrhoeae, and P. aeruginosa), with efficiencies of 118, 120, and 124 %, respectively. The presence of f-MWCNTs in the tested samples is responsible for the observed high antibacterial activity of the two hybrids. MWCNTs have been shown to have strong inhibitory effects on a variety of bacteria in previous work [49].
All postulated mechanisms to explain the bactericidal effect of CNTs in general are not fully known, and there are numerous factors that influence its antibacterial action. The diameter, electrical structure, residual catalyst, length, surface functional group, and other parameters are among them. [50]. In terms of the functionalized MWCNTs employed in this investigation, it has been noted that functionalization is a good way to improve their dispersion in different matrices while also increasing biocompatibility and lowering toxicity in human cells [51]. The optical density of E. coli was significantly reduced when amine or carboxyl moieties were added to MWCNT. According to certain observations, the functionalized carboxylated MWCNTs increased membrane roughness and hydrophilicity, which made them more resistant to bacterial adherence. These findings suggested that f-MWCNT surfaces could be used in the manufacture of medical devices and biomedical applications [52-53].
The incorporation of MWCNTs or f-MWCNTs into chitosan or other synthetic polymers to obtain potentially helpful antibacterial surfaces against Gram-positive and Gram-negative bacteria has been investigated. This could lead to the development of unique physicochemical properties for these polymeric substrates, as well as the prospect of using these composites in a variety of biological applications such as tissue engineering, biosensing, wound dressing, and drug administration [54-55]. Figure 7 illustrates the antibacterial efficiency of the investigated samples with respect to the standard reference drug.
Table 2: The antimicrobial activities of the investigated samples against some G +ve and G -ve bacterial strains
Sample
|
Inhibition zone diameter (mm/mg sample)
|
Bacillus Subtitles
|
Staphylococcus aurous
|
Streptococcus faecalis
|
Escherichia coli
|
Neisseria gonorrhea
|
Pseudomonas gonorrhoeae
|
|
G+ve
|
|
|
G-ve
|
|
DMSO
|
0.0
|
0.0
|
0.0
|
0.0
|
0.0
|
0.0
|
Ampicillin (100µg/mL)
|
20
|
18
|
18
|
22
|
20
|
17
|
Cs
|
13
|
15
|
12
|
14
|
11
|
12
|
Cs-g-PVP
Cs-g-PVP/MWCNTs (3%)
Cs-g-PVP/MWCNTs (5%)
|
17
19
20
|
17
19
21
|
15
17
17
|
19
25
26
|
17
22
24
|
16
18
21
|