Chitosan/PVA/PEG Blend Strengthened with MgO Nanoparticles for Antibacterial Application

Chitosan holds net ionic positive charges, which contribute its ability to chemically bind with negatively charged fats, lipids, metal ions, proteins, and microorganisms. Magnesium oxide (MgO) nanoparticles are important inorganic materials with a wide band-gap used in many applications such as catalysts, antibacterial and medical products. The aim of this study was to investigate the effect of Chitosan (CHT) hydrogel loaded MgO nanoparticles on the bacterial growth. CHT/ poly vinyl alcohol (PVA)/ poly ethylene glycol (PEG) hydrogel was blended with various amounts of MgO nanoparticles. The surface morphology of the obtained blends was investigated by Field Emission Scanning Electron Microscope (FE-SEM). Evidently, surfaces with appropriate roughness were obtained for most of the prepared hydrogels. Fourier Transform Infrared Spectroscopy (FT-IR) and Energy Dispersive X-Ray analysis (EDX) were also included in this paper. Thermal properties of all samples was studied by DSC-TGA curves. The antibacterial activity of the prepared hybrids CHT/PVA/PEG/MgO nanoparticles have performed against gram positive bacteria Staphylococcus aureus (S.aureus) and Streptococcus, as well as gram negative bacteria Pseudomonas aeruginosa (P. aeruginosa) and Escherichia coli (E.coli). In this study, MgO nanoparticles various proportions presented high efficiency towards gram positive microorganisms. High resistance of gram negative bacteria against the final products was extremely detected according to measured inhibition zones which were between (0-9) mm.


Introduction
An integration of inorganic-organic, inorganic-inorganic, and organic-organic materials is adopted to fabricate hybrid composites, recently. Utilizing of natural polymers like chitosan loaded inorganic metals or metal oxides nanoparticles has attracted the attention of researchers to engineer hybrid composites [1]. Chitosan is a natural positive charged polysaccharide [2], which attracts researchers' interests due to its unique properties including biocompatibility, biodegradability, non-toxicity, hydrophilicity, and anti-bacterial activity [3][4][5][6][7][8]. These characteristics have made chitosan a particularly beneficial compound in a wide range of applications, for instance, medical, chemical, environmental and agricultural fields, [9] as well as, in wound healing biomaterials, and chelating factor due to its capability to combine with proteins, cholesterol, fats, and metal ions [10]. Chitosan has a rigid crystalline structure owing to intra-molecular and inter-molecular hydrogen bonding which introduces its polymorphism [11]. Chitosan is unsolvable in many solvents but is soluble in dilute organic acids, for example, acetic acid, succinic, formic, lactic acids of pH < 6.0 due to primary amino groups on chitosan backbone which considerably produce a strong base [12]. Blended polymers fabrication has become one of the convenient methods used to provide polymeric materials with desired properties for practical applications. In particular, chitosan blended with (PVA) has been stated to have good mechanical and chemical properties, [13] through hydrophobic branch chain aggregation and hydrogen bonds formation [14]. (PEG) has also been widely utilized because of its biocompatibility and non-toxicity. Chitosan/PEG hydrogel can swell significantly due to alterations in the pH of the medium and as response to the positive charges on the network [15]. Hydrogels are formed by polymer networks of cross-linked sides that have an excessive amount of hydrophilic functional groups with affinity for water [16,17]. Many diseases like lung infections, gingivitis, and otitis media can be resulted from various bacterial pathogens. Nanomaterials have the ability to struggle bacterial growth. Resistance development of bacteria can be inhibited by several antibiotics encapsulation techniques within nanomaterials [18][19][20]. Antibacterial drugs applied to infectious region through nanomaterials enhance driving the medication to the diseased cells with low side effects to sick people. High molecular weight CHT and low molecular weight CHT doped nanomaterials have presented high efficiency in combating gram-positive and gram-negative bacteria, respectively [21,22]. Chitosan antimicrobial activity involves two main mechanisms. The first mechanism expected that cations on chitosan chains could bind with negative charges at the bacterial cell wall, which affect permeability and lead to penetrate outside solutes inside the cells. The second explanation suggested that it could interact with Deoxyribonucleic acid (DNA) of bacterial cell and then prevent Ribonucleic acid (RNA) synthesis [23]. In this work, CHT nanopowder/ PVA/ PEG hydrogels were hybridized with different weights of nanosized magnesium oxide MgO powder. FT-IR spectrum, FE-SEM, EDX, DSC-TGA analysis, and antibacterial efficiency were collected to study the active functional groups, surface morphology, thermal stability, and bacterial lethal efficiency of the blends.

Materials
Chitosan nanopowder which produced by Micxy Chemical Co. with a degree of deacetylation (DD) of 95.7% and about 80 nm particles size was used. PVA (average molecular weight is 67000 g mol -1 ) and PEG (average molecular weight is 3500 g mol -1 ) were also utilized as plasticizer polymers. MgO nanoparticles (<50 nm) with high purity >99% were bought from Chinese source. Acetic acid glacial (extra pure/ MW. 60 g mol -1 ) produced by Tomas Baker/India also used as solvent with chitosan. Distilled water was required as solvent and for cleaning the used tools. Gram positive and gram negative species of (S.aureus, Streptococcus), and (P. aeruginosa, E.coli), respectively are selected to examine the antibacterial effect of the prepared samples.

Preparation of the Main Polymeric Solutions
Separated steps were followed to prepare CHT, PVA and PEG solutions, firstly. 1.5g of chitosan nanopowder was dissolved in 100ml distilled water with the addition of 2ml acetic acid (2% concentration) which is a good solvent for chitosan. The mixture was well stirred at room temperature and moderate speed for about 30 min by using a magnetic stirrer. A clear and homogeneous solution that optically seen and kept away into a sealed container. 5g of each PVA and PEG polymers were dissolved into 100ml distilled water, individually. To produce a 5% concentration of each agent; the PVA solution was prepared at 80℃ for 15min stirring, whereas PEG solution was mixed by a sonicator bath at room temperature for 5min and then saved into sealed containers.

Hydrogel Synthesis
In this study, CHT/PVA/PEG doped MgO nanoparticles were blended to investigate their role as antibacterial product. A sensitive electronic balance was used to measure various amounts of MgO (0.04, 0.06, and 0.08) g. Put (0.04g) of MgO in a plastic beaker and then adding (1ml) of PVA solution with well shaking by magnetic stirrer for (1min), next that, (1ml) of PEG solution poured to the mixture with continuous stirring for (1min), also. When a clear white blend was observed, (5ml) of the previously prepared chitosan was gradually added (drop by drop) by a burette and the final product was left under continuous stirring for (30 min). The same steps were exactly managed for the two other amounts of MgO. Three individual hydrogel samples were kept into open tubes to let them dry at room temperature for a week. Semi-gel appeared at the end of the week and then dividing each sample into small specimens for characterization analyses. FE-SEM, EDX, FT-IR, DSC-TGA and antibacterial results were accomplished.

Thermogravimetric Analysis (DSC-TGA)
DSC-TGA analyses were carried out with a Universal V4.5A instrument by applying Ramp method. Approximately, (3-6) mg of provided samples were dual cured within  ºC in air at 20 ºC/min heating rate.

Incubation of the Selected Bacteria
The antibacterial activities of the prepared gels were investigated against E.coli, P.aeruginosa, S.aureus, and Streptococcus microorganisms. A 100 μl of culture suspension of the tested bacteria, roughly 10 6 CFU (colony forming units), was dispersed onto agar surface. Well diffusion method on Mueller Hinton agar (MHA) was employed to examine the microorganism growth. Concerning the antibacterial activity of the produced samples, 500 μl of each gel was put into wells of (6 mm depth, 6 mm diameter) previously made on the agar. Inhibition zones were shown after 24 h at 37 °C of incubation conditions. The growth of bacteria around wells was recorded according to the diameter of inhibition zones. Inhibition zone is the area surrounding the wells that filled by the prepared samples with different zone diameters depending on the antimicrobial efficiency.

FT-IR analysis
The FT-IR spectra were obtained within the range of 400-4000 cm −1 , but the major fingerprints of the tested samples, as shown in Figure 1, are between (1000-3345) cm −1 . The infrared spectrum of pure chitosan (Figure 1) reveals around 894 cm -1 and 1159cm -1 peaks pointed to saccharine structure, and a weaker amino characteristic peak appears at around 1597cm -1 . The peak at 1260cm -1 is the absorption of δ (O ̶ H) refers to bending state, and the weak band at 1381cm -1 has been assigned to the -CH3 symmetrical deformation mode. The broad characteristic peak in the region 3100-3600 cm −1 , related to the stretching vibration of -CH, -NH2 and -OH bonds. The peaks at 1640 cm −1 for -NH2 and 1597cm -1 for ̶ OH are signed to bending vibrations on CHT backbone [24]. PVA scheme ( Figure 1) shows transmittance bands at 2937, 1439, 1257, 1092 and 843 cm -1 , which are assigned the Ʋa (CH2) asymmetric stretching, δ(CH ̶ OH) symmetric bending mode, γw(CH) wagging mode, Ʋ(C ̶ O), and Ʋ(C ̶ C) resonance, respectively [25]. Finally, the wide recognized band around 3421cm -1 was due to the stretching vibration of O-H bonded to N-H [26]. The characteristic band at 1100 cm -1 of PEG (Figure 1) was attributed to the bending vibration of C-O, and sharp bands at 2900 related to asymmetric stretching vibration of sp 3 C-H [24]. The disappearance of crystallization sensitive bands of pure chitosan at 1078 cm -1 in the blends (Figure 1) implying that an intermolecular interaction between PVA/PEG with CHT disrupts the crystallinity of chitosan at the blend status, and also a wide-deep band around 3500 cm -1 related to the stretching vibration of amine and -OH groups with implicit -CH (sp 2 ) bonding [24]. CHT/ PVA/ PEG/ MgO hybrids ( Figure 1) were showed broad percentage transmittances greater than 3300 cm −1 due to the stretching vibrations of the O-H and N-H bonds. For hybrid CHT, all the composites showed higher percentage transmittances at wavenumbers below 2000 cm −1 compared with the pure chitosan. The peak at 2857cm −1 , symmetric stretching of C-H in pure CHT, was doublet in MgO/CHT blends nearby to the first one at about 2940cm -1 referring to asymmetric C-H stretching bond. OH and -NH2 stretching mode at (3360-3500) cm −1 appeared wider and less intensity than that at the spectrum of pure CHT. -OH bending mode on the CHT backbone has shifted to lower frequencies around (1565-1573) cm −1 for patterns (E, F, and G) as it can be bonded with Mg +2 . Moreover, the appearance of peaks around 850 and 925 cm −1 on the spectrum of CHT/Mg gels is a characteristics of the stretching vibrations of Mg-O and Mg-N with different intensities [27].

FE-SEM/ EDX analysis
SEM is a valuable tool to evaluate the scaffold characteristics (pores, particles size, and surface morphology) of prepared hydrogels. Figure 2(A, B, C) shows the FE-SEM micrographs from the hydrogel CHT/PVA/PEG loaded (0.04, 0.06, 0.08) g of MgO nanoparticles, respectively. Images A and C display porous surfaces with many encapsulated features related to ability of chitosan for wrapping when cross-linked with PVA and PEG polymers. However, image B did not show porous micro-structures, but it shows, to some extent, a smooth texture with granular shapes instead. This probably due to the prepared blend homogeneity or due to formation of H-bonds, which led to the irregular polarity in the internal system [28]. This reason is in agreement with the results obtained from FT-IR patterns in Figure F and EDX peaks in Figure 3. Herein, it could be observed that samples of welldeveloped open pores can facilitate mass transfer of loaded nanoparticles for particular applications, [29] as it will be evidenced in the next section.

Thermal Properties of the Hydrogels
Thermogravimetric analyses TGA of the three prepared hydrogels were relied to study their thermal stability, as shown in Figure 4. The three tested composites of CHT/ PVA/ PEG/ MgO (0.04, 0.06, and 0.08) g showed a significant weight change of about 5% between (25-160) ºC, which could be proportional to evaporate the residual moisture included in the hybrids network as agree with other research studies [30,31]. The thermal degradation started at higher temperatures due to weight loss over time. The first two diagrams (A, B) showed fairly same three stages of decomposition process appeared at about (220, 340, and 470) ºC with an approximate weight remain of (0.8-1) g at the end of this stage, respectively. For the third diagram (C), the three steps are still observed but with a notable shifting to higher temperatures. The new thermal behavior could be an indication that increasing MgO amount to 0.08g led to rise the degradation point to around 550 ºC with great ratio of remaining weight 52% wt. The previous important degradation steps can be attributed to dehydration, deacetylation, depolymerisation of utilized polymers [32], and MgO nanopowder decomposition. An obvious decrease in weight loss was detected in CHT/ PVA/ PEG/ MgO nanocomposite hydrogels with increasing MgO nanopowder quantity. It is an indication of improving their thermal stability to be optimal antimicrobial and medical products [33].

Bacterial Investigations
The antibacterial assay of the prepared nanocomposites is stated in Figure 5 and Table 1. Figure 5 showed remarkable changes in the diameter of the inhibition zones surrounding the determined wells. This may be related to the ability of CHT blend and MgO nanoparticles in attacking cell-walls of the bacteria and restricting their activity. It is well known that chitosan has tremendous metal-binding abilities, where amino charged groups interact with metals [34]. The interaction between amino groups and divalent ions like Mg 2+ prevents the production of toxins and inhibits bacterial growth [35]. The antibacterial effect of MgO may result a damage to the peptide linkages at super-oxide ions on the carbonyl group, which causes degradation the protein of the pathogens. MgO reacts with intracellular oxygen to create reactive oxygen species ROS such as O2 •-, OH • and H2O2 which could be delivered into the medium [36] and then motivates the destructive efficiency towards the bacterial cell wall [37]. The results revealed that the CHT hybridized with polymeric agents and MgO nanopowder did not exhibit a significant antibacterial efficiency against gram negative strains as compared with gram positive ones. Thereby, slightly inconsiderable inhibition zones for all tested samples against P.aeruginosa and E.coli between (0-9) mm were observed. In agreement with  who reported that MgO nanoparticles exhibited high efficiency against gram positive than the gram negative microorganisms. This behavior may be related to the susceptibility differences of various grown bacteria tested in this work [38,39]. Principally, gram-positive bacteria have a single lipid bilayer (monoderms), whereas gram-negative bacteria have two bilayers (diderms) [40]. Generally, nanoparticles could penetrate the cell wall by either diffusion through the cell membrane or inserting into the nucleus which cause mitosis as the membrane dissolves during this process [41]. The fluctuated values of measured diameters of the inhibition zones for the prepared hybrids, as appeared in Table 1, could be also attributed to various surface morphology of examined samples. As shown in Figure 2, major characteristic structures such as: pores, size and shape of features, distribution of MgO nanoparticles along the active area, cracks, and bulk grains formation may have a significant role in the antibacterial mechanism.

Conclusions
Blended hydrogel samples of CHT/PVA/PEG with various proportions of MgO nanoparticles were prepared. Investigation of the surface morphology of the obtained blends displayed good porous texture for only two MgO concentrations (0.04 and 0.08) g, while the last sample showed a smooth and bulk grains surface as revealed by the results of FE-SEM images. From FT-IR schemes, disappearance of bending amino band for 0.06g MgO sample may reflect the lack of its antibacterial effect, and its non-porosity could be explained due to the intermolecular hydrogen bonding existing between the amino groups of chitosan and the hydroxyl groups of PVA/PEG. The antibacterial activity of the prepared hybrids have executed against gram positive bacteria (S.aureus, Streptococcus) and gram negative bacteria (P.aeruginosa, E.coli). In this study, the added MgO nanoparticles performed more efficient against gram positive microorganisms than the other type gram negative. The differences observed in the diameter of inhibition zone may be due to the variation in the susceptibility of diverse pathogens to the prepared nanoparticles. Characteristically, gram positive bacteria have no external membrane in the cell wall. Otherwise, gram-negative bacteria have more complex cell wall structure with an interfacial layer of peptidoglycan between outer and cytoplasmic membranes. Accordingly, the cell wall of gram positive bacteria can be easily degraded. The antibacterial efficiency of CHT/MgO nanoparticles gel may be also responsible to the binding of surface oxide ion to the bacteria and depends on the available surface area for interaction.