Synthesis and Characterization of Grafted Chitosan Blending with Polyvinyl alcohol / Nanocomposite and Study Biological Activity

: The study of biopolymers and their derivative materials had received a considerable degree of attention from researchers in the preparation of novel material. Biopolymers and their derivatives have a wide range of applications as a result of their bio-compatibility, bio-degradability and non-toxicity. In this paper, chitosan reacted with different aldehydes(2,4 –dichloro-benzaldehyde or 2-methyl benzaldehyde), different ketones (4-bromoacetophenone or 3-aminoacetophenone) to produce chitosan schiff base (1-4) . Chitosan schiff base (1-4) reacted with glutaric acid or adipic acid in acidic media in distilled water according to the steps of Fischer and Speier to produce compounds (5-12) respectively. Grafted chitosan (5-12) blended with synthetic polymer PVA to produce compounds (13-20) , then these polymers were blended with nano Ag by using a hotplate stirrer for 60 min to produce nanocomposites. The synthesized polymers were identified via spectral analysis techniques, including FTIR , 1 H-NMR and scanning electron microscope (SEM). Finally, studied anti-bacterial activities of some of the prepared polymers.


Introduction:
Chitosan has a wide-field in biological activities such as antimicrobial, antitumor and used pharmaceutically as an anticoagulant agent 1 , also can be useful in several fields such as textiles, environmental protection, water treatment, cosmetics and bio-technology due to its ability to produce bifunctional materials [2][3][4] .
The structure of chitosan is easy to be modified to several derivatives due to -OH and NH2 groups found 5 which distinct chitosan from cellulose 6 . Amino group can be condensed with the carbonyl group of aldehydes or ketones. The modified chitosan exhibits new properties such as solubility, biological activity 7 , biocompatibility and hydrophilicity 8 .
Chitosan's anti-bacterial characteristics have been associated with its poly-cationic character [9][10][11] . The protonated functional groups of the chitosan interacted with the negatively charged micro-organism cell membranes, leading to damage and, ultimately, eradication. On the other hand, those characteristics have been highly dependent upon basic bio-polymer characteristics, like deacetylation degree or molecular weight, in addition to external conditions where material has been utilized 12 . Presently, the interest of scientists' has been increasingly focused on the use of the biodegradable polymer materials 13 . The chitosan (Ch) and Poly(vinyl alcohol) (PVA) 14 have been considered as best known environmentally friendly polymer types , both of them, and the materials that have been based upon them, found numerous application areas in the pharmaceuticals, medicine, and materials coming in contact with the food, this is particularly a result of their bio-compatibility, bio-degradability, and low or even full lack of the toxicity 13 . The ways by which those polymers have been modified mainly contribute to the enhancement of the materials' characteristics 9 . In cases of the PVA and chitosan, their sufficient miscibility results from bonds of hydrogen that are formed between their functional groups. This is why, the blending of the Chitosan with the PVA plays a role in receiving homogeneous materials that have anti-microbial characteristics and more sufficient mechanical characteristics compared to the Chitosan 15 .The purpose of this work was to prepare new nanocomposites with good biological activity. Resulting from blending grafted chitosan with polyvinyl alcohol and silver ions.

Materials:
The chemicals were supplied from BDH, CDH and SCR.

Instrumentation:
The FT-IR spectra have been registered on Shimadzu FTIR8400-s, ranging between 400cm -1 and 4,000cm -1 , using the potassium bromide disk. 16 Chitosan (0.5 g.) has been dissolved in glacial acetic acid and ethanol (15mL) with stirring for a period of 30 min at the temperature of the room. After that , variety of aromatic aldehydes (2,4 dichloro benzaldehyde or 2-methyl benzaldehyde) or different ketones(4-bromoacetophenone or 3aminoacetophenone) were added to the mixture .The mixture has been stirred magnetically then heated at 60°C for 24h., followed the cooling, the crude product has been washed using ethanol. The product has been dried at room temperature. 17 Polymers (5-12) were synthesized from (0.5 g.) of compounds (1-4) was hung in 25 mL of H2SO4 (2M), then added (glutaric acid or adipic acid) into the solution, after that has been refluxed for a period of 6h. followed by cooling to r.t. pH value has been settled to 7 through the neutralization with the sodium bi-carbonate, and after that it has been precipitated in the acetone, followed by filtering, cleaning by acetone, then drying at a temperature of 60 o C in the oven. As shown in schemes (1), (2). Synthesis of Polymers Blend (13-20) 18 Polymers blend were produced by using the solvent casting method. The solutions of the grafted chitosan (5-12) were produced by dissolving (5-12) in a 2% solution of aqueous acetic acid with stirring at the temperature of the room. Polyvinyl alcohol (PVA), figure 1 was dissolved in the hot water for the purpose of producing 5 wt% solutions of polymer. Both solutions of the polymers were mixed and a homogenous solution has been made with the use of a hot-plate stirrer for a duration of 60min. the Grafted Cs/PVA blends have been done through the mixing of (one ratio) Grafted Cs: PVA (5:5).

Figure 1. Structure of Polyvinyl alcohol(PVA)
Preparation of Grafted Cs/ PVA Silver Nanocomposites 19 100mg of the dried Grafted Cs/PVA blend has been put in 50mL of the silver solution of a 250mg/L concentration as well as 1.5h sonication to bond the silver nanometal in blend matrix by the electro-static force.

Results and Discussion:
Chitosan Schiff base has been produced from the reaction of the chitosan with the ketone or aldehyde in the ethanol and glacial acetic acid. The FTIR spectrum of compound(1), had shown a new band of absorption at 1651cm -1 that has been attributed to the (C=N) of the imine group. The band of the absorption at 1595cm -1 that has been assigned for C=C of aromatic aldehyde 20 (13-20) were prepared research of characteristics of obtained blends had shown a good level of the miscibility between the PVA and Chitosan that had been shown by FT-IR results of the compound (13), the band broadening in (2400-3600)cm -1 region because of a strong inter-molecular bonding of hydrogen that exists between amino groups of Chitosan and PVA's hydroxyl groups, 1635cm -1 as a result of (C=N) and 1708 cm -1 which meaning C=O ester group 22 , and for the other polymers, the band values are illustrated in table 3.   Scanning electron microscope studies (SEM) 23 roughness to blend membrane (Grafted chitosan /PVA). Adding PVA results in the alteration of the blend membrane surface topography and has a considerable impact on the cell spreading. SEM micrograph for presence of AgNPs has been noticed to be with the homogenous distributions on the matrix surface.
The average nano size of the particles is ranged between ( 64 -12 ) nm for Ag nanoparticles.
The SEM images revealed that there were significant changes on the surface of prepared blends after interaction between the polymers. Nanoparticles are in a homogeneous distribution over the matrix's surface. The particles in nanocomposite film were found with almost spherical morphology. However, some of the agglomerations of nanoparticles were also found in the figures and the surface was somewhat rough. Biological activity 25 Biological activities of the Grafted Chitosan , Grafted chitosan Blended with PVA , polymer blend of PVA / grafted Chitosan with silver nanocomposite, have been tested against two pathogenic bacteria types (G+) Staphylococcus aureus and E. coli (G-), utilizing the Diffusion inhibition approach and compare with(Ag) nano. The results of antimicrobial activity are represented in Table 4, Figure 5. When compared to Ag nanocomposites, the ternary mix (PVA / Grafted Chitosan) with Ag nano composite that was exhibited good antimicrobial activities.
For Ag nanocomposite the silver exhibits antibacterial properties which lead to biomedical applications. Silver's antibacterial action is based on Ag + , binding tightly to the electron donor groups in microbial cell walls such as sulfur, nitrogen or oxygen. Ag ions work through the displacement of other important metal ions like Ca 2+ and Zn 2+ . Ag nanoparticles have complex impacts on bacterial cells 26,27 . However, the impact of silver nanoparticles on bacterial cells is mediated in a variety of ways 28 .The following are some of the mechanisms that have been summarized and presented : (i)the capacity of silver nanoparticles to bind to and enter the bacterial cell wall 29 , (ii) the production of free radicals by Ag NPs, which may damage and porous the cell membrane 30 , (iii) NPs can release Ag ions, which can bind and inactivate thiol groups of several important enzymes 31 and (iv) nanoparticles can alter signal transduction in bacteria, preventing bacteria from growing 32-34 .