236 This work is licensed under a Creative Commons Attribution 4.0 International License IHJPAS. 37 (1) 2024 Ibn Al-Haitham Journal for Pure and Applied Sciences Journal homepage: jih.uobaghdad.edu.iq PISSN: 1609-4042, EISSN: 2521-3407 1Widad Abed Shlaka 2Ruwaidah Samir Saeed* 3El-Sayed Negim 1Department of Chemistry, College of Science, Mustansiriyah University, Baghdad, Iraq. 2Department of Chemistry, College of Education for Pure Science Ibn Al-Haitham, University of Baghdad, Baghdad ,Iraq. 3School of Petroleum Engineering, Satbayev University, 22 Satpayev Street, Almaty 050013, Kazakhstan. *Corresponding Author: ruaida.s.s@ihcoedu.uobaghdad.edu.iq Abstract In the present study, synthesis of bis Schiff base [I, II] by reaction of one mole of terephthalaldehyde with two mole of 2-amino-5-mercapto-1,3,4-thiadiazole or 4-amino benzene thiol in the ethanol absolute, then compounds [I,II] were reacted with Na2CO3 of distilled H2O, then chloroacetic acid was added to yield compounds [III,IV]. O-chitosan derivatives [V,VI] were synthesized by reaction of chitosan with compounds [III,IV] in acidic media in distilled water according to the steps of Fischer. O–chitosan (grafted chitosan) [V,VI] was blended with synthetic polymer polyvinyl alcohol (PVA) to produce polymers [VII,VIII], then these polymers were blended with nano: Gold or Silver by using a hotplate stirrer for 3 hours to produce nanocomposites [IX- XII]. The synthesized polymers were identified using spectral analysis techniques, including FTIR,1H-NMR, and scanning electron microscope (SEM). Molecular docking was studied, where operations are used to predict the binding status of compounds with the enzyme and to calculate the free energy (ΔG) of the prepared compounds. Finally, the study of biological activities was screened via two types of bacteria. Also, the anti-cancer activity against human lung adenocarcinoma cells (A549) was studied and compared with standard cell line [REF(R7540) Rat Embryonic Fibroblasts] of some of the blended polymers and nanocomposites, then the acute toxicity test of some nanocomposites was performed. Keywords: Rat embryonic fibroblasts, Nanocomposites, Molecular docking, Toxicity study, O- Chitosan 1. Introduction Chitosan is produced from chitin found in the cell walls of fungi, the cuticles of insects, and the shells of mollusks and crustaceans [1]. Chitosan can be defined as a cationic linear polysaccharide made up of specific N-acetylglucosamine units and (β 1,4) linked glucosamine units [2]. Since Received 12 March 2023, Received 16 April 2023, Accepted 2 May 2023, Published 20 January 2024 Synthesis and Study Medical Application of Nanocomposites Based on Grafted Chitosan /Polyvinyl Alcohol doi.org/10.30526/37.1.3327 https://creativecommons.org/licenses/by/4.0/ https://jih.uobaghdad.edu.iq/index.php/j/index#1609-4042 https://jih.uobaghdad.edu.iq/index.php/j/index#2521-3407 mailto:ruaida.s.s@ihcoedu.uobaghdad.edu.iq https://orcid.org/0009-0009-3512-885X mailto:Wdadshlaka@gmail.com https://orcid.org/0000-0002-8900-1557 mailto:ruaida.s.s@ihcoedu.uobaghdad.edu.iq https://orcid.org/0000-0002-4370-8995 mailto:elashmawi5@yahoo.com IHJPAS. 37 (1) 2024 237 chitosan is unique in that it possesses qualities including biocompatibility, non-toxicity, anti- microbial activity, anticancer activity, and biodegradability, it could be employed effectively in a variety of biomedical applications [3,4]. The structure of chitosan is easy to modify to several derivatives due to -OH and NH2 groups found [5], which distinct chitosan from cellulose [6]. The modified chitosan exhibits new properties such as solubility, biological activity [7], biocompatibility, and hydrophilicity [8]. Poly (vinyl alcohol) (PVA) can be defined as one of the synthetic polymers, which are mainly composed of C-C bonds [9,10]. It's non-toxic, water-soluble, biocompatible, and biodegradable, which makes it widely applicable in the biomedical field [11]. In the case of the PVA and the chitosan, their sufficient miscibility results from bonds of hydrogen that are formed between their functional groups [12,13]. Chitosan blend with PVA plays a role in receiving homogeneous materials that have anti-microbial characteristics and are more sufficient than Chitosan [14,15]. The biomedical field has paid close attention to gold nanoparticles (AuNPs), among other functional nanomaterials, because of their nontoxicity, biocompatibility, and ease of manufacture [16,17]. Additionally, silver nanoparticles (AgNPs) have been shown to have enough potential to treat cancer [18]. The physical, chemical, and biological properties of CS, when combined with AuNPs, AgNPs were widely utilized in various medicinal applications and drug delivery [19,20]. 2. Materials and Methods BDH and SCR supplied chemicals. The 1H-NMR spectra were performed by the company Ultra Shield 500 MHz, Bruker, University of Tehran, Iran, and DMSO as solvent has been used- Shimadzu FT-IR-8400 s, with FT-IR spectra between 400 cm-1 and 4000 cm-1. A fully licensed CCDC genetic optimization for ligand docking (GOLD) Hermes 2021.2.0 (Build 327809) was used to record the molecular docking studies for the compounds and visualize the protein, ligands, hydrogen bonding interactions, short contacts, and bonds length calculation. University of Tabriz, Iran, performed SEM. 2.1 Synthesis of 2-amino-5-mercapato-1,3,4-thiadiazole According to the literature [21], this compound is prepared and gives a good yield of 87%, M.P. (229-231) Celsius. 2.2 Synthesis of compounds [I,II] Terephthalaldehyde (1.34 g, 0.01 mol) mixed with (2.5 g, 0.02 mol) 4-amino benzene thiol or (2.66 g, 0.02 mol) 2- amino-5- mercapto -1,3,4 – thiadiazole, Ethanol absolute (20 mL), with two drops of glacial acetic acid, refluxing at 70 °C for 18h. The reaction mixture was cooled, and the yellow precipitate filtration was recrystallized from ethanol and dried to yield 80% and 95% [22]. [III,IV] 2.3 Synthesis of compounds One of the compounds (0.01 mol) [I, II] was mixed with (0.04 mol) Na2CO3 in (15 mL) of distilled H2O, then (0.02 mol) of ClCH2COOH was added. The solution refluxed for six hours, then was added conc. HCl reached out PH= 2. then filtered and washed with H2O and recrystallized by EtOH [23] Scheme 1. IHJPAS. 37 (1) 2024 238 2.4 Synthesis of O- chitosan derivatives [V,VI] Chitosan (0.5 gm) was hanging in 25 mL of H2SO4 (2M) and added (0.01 mol) of compounds [II I, IV] to this solution. The mixture refluxed for eight hours before cooling. NaHCO3 was used to neutralize the pH and keep it at 7. The product was precipitated in acetone, filtered, and then was hed with acetone to remove any remaining acid. It was then dried at 60 Celsius in an oven for 24 hours [24]. 2.5 Synthesis of polymer blend [VII- VIII] Polymer blends were produced by using the solvent casting method. The grafted chitosan [V, VI] solutions were dissolving [V, VI] in a 2% solution of aqueous acetic acid with stirring at room temperature. Polyvinyl alcohol (PVA) was dissolved in the hot water to produce five wt% polymer solutions. Both solutions of the polymers were mixed, and a homogenous solution was made using a hot-plate stirrer for 60 min. The Grafted Cs/PVA blends were done through the mixing of (one ratio) Grafted Cs: PVA (5:5) [25]. 2.6 Synthesis of grafted Cs/ PVA /nanocomposites [IX- XII] About 100 mg of the dried Grafted Cs/PVA blend [VII- VIII] was put in 50 mL of the Au or Ag solution of a 250 mg/L concentration by using a hotplate stirrer for (3 hr.) to bond the gold and silver nanometal in blend matrix [26]. Scheme 1. Synthesis of compounds [I- IV] IHJPAS. 37 (1) 2024 239 Scheme 2. Synthesis of O-Chitosan [V,VI] Scheme 3. Synthesis of blend polymers and nanocomposities [VII -XII] 3. Results and Discussion 3.1 The FTIR and 1H-NMR of synthesis compounds, grafted chitosan and blend polymers The synthesis of new derivatives started with bis Schiff bases is demonstrated in Scheme 1. Compounds [I, II] were created by reacting terephthalaldehyde with either 4-amino benzene thiol or 2-amino-5-mercapto-1,3,4-thiadiazole in ethanol at reflux for 18 hours. The compound's FT-IR [I] revealed appearance bands at (2547 and 1639) cm-1, respectively, because of the SH group and (C=N) as shown in Table1. Compounds [III, IV] were prepared in primary media by the reaction compounds [I, II] with chloroacetic acid in distilled water. The FTIR spectrum of compound [III] in Table 2 establishes a band at (3400-2400) cm-1 for the hydroxyl group and (1688) cm-1 for the carboxylic group. The 1H-NMR exhibited a broad singlet signal with a chemical shift at δ 13.14 ppm as a result of the two protons of carboxylic protons; additional signal at δ10.07 ppm due to the presence of two protons for CH-N, multiple peaks appeared at δ(7.33-8.89) ppm for aromatic protons as well as a singlet signal at δ 3.91 for four protons for S-CH2. The reaction between [III, IHJPAS. 37 (1) 2024 240 IV] and chitosan in distilled water in acidic media, Scheme (2), produced the new O-chitosan derivatives [V, VI]. The FT-IR of polymer [V] in Table 3 interpreted with the existence of a large band at (3294) cm-1 as the stretch band regarding O-H as well as N-H from intra- and extra- molecular hydrogen bonding of chitosan molecules as well as a new absorption band at (1716) cm-1 due to ester's C=O. The 1HNMR of polymer [V] elucidated a singlet signal with a chemical shift region at δ 12.75 ppm as a result of the proton of carboxylic protons (8.72-8.75) ppm due to the presence of protons of hydroxyl groups of chitosan, a multiple signals at δ(7.20-8.10) ppm for aromatic protons, singlet signal at δ7.98 ppm as a result of the presence of two protons for N-CH groups, singlet signal at δ6.53 ppm for proton of SCH2 groups and signal at 5.84 ppm due to CH2O, also the characteristic region at (3.42-4.32) ppm corresponded to the non-anomeric proton (H-1, H-3, H-4, H-5 and H-6) of chitosan, singlet signal at δ1.89 ppm for two protons of NH2 group [27], a signal at 1.20 ppm assigned to H-2 and signal at 1.02 ppm existed because of the presence of CH3 of N-alkylated of glucosamine residue. O-Chitosan derivative (Grafted chitosan) blended with PVA [VII- VIII] was prepared research of characteristics of obtained blends had shown a good level of the miscibility between the PVA and Chitosan that FT-IR results of the polymer had demonstrated [VII], the band broadening in (2400-3600) cm-1 region because of a solid inter- molecular bonding of hydrogen that exists between amino groups of Chitosan and PVA's hydroxyl groups, 1715 cm-1 which meaning C=O ester group. Table 1. The FT-IR spectroscopy data of compounds [I,II] Comp. No. (S-H) cm-1 =C-H arom. (C=N) (C=N) of thiadiazole (C-S) [I] 2547 3050 1639 - 696 [II] 2540 3081 1645 1613 700 Table 2. The FT-IR spectroscopy data of compounds [III,IV] Comp. No. (O-H)cm-1 (C-H)arom. cm-1 (C=O) carboxlic cm-1 (C=N) (C=C) cm-1 [III] 3400-2400 3055 1688 - 1583 [IV] 3400-2400 3043 1690 1607 1595 Table 3. The FT-IR of grafted chitosan and blend polymers[ [V-VIII] Comp. No. υ (O-H) and (N-H) υ (C-H) aliph. υ (C=O) ester. υ (C=C) υ (-CH2-O-CO) υ (C-O-C) [V] 3294 2938, 2914 1716 1579 1371 1069 [VI] 3419 2943, 2872 1710 1590 1243 1048 [VII] 3274 2909, 2858 1715 1601 1271 1068 [VIII] 3269 2939, 2909 1703 1580 1240 1080 3.2 Molecular docking study Molecular docking in Table 4, Figure 1, and Figure 2 were studied, where operations are used to predict the binding status of compounds with the enzyme and to calculate the free energy (ΔG) of the compounds prepared with the enzyme 3-hydroxy-3-methylglutaryl-CoA reductase (HMGR), as well as the study of molecular similarity. 1DQ9: Mevalonate was produced due to 3-hydroxy-3-methylglutaryl-CoA reductase (HMGR), a committed step in biosynthesis regarding isoprenoids and sterols. The activity related to HMGR is regulated through degradation, synthesis, and phosphorylation to keep the level of mevalonate- derived products stable. The human enzyme was successfully targeted through drugs in the clinical IHJPAS. 37 (1) 2024 241 therapy of excessive serum cholesterol levels, along with the physiological regulation of HMGR. The catalytic portion of human HMGR in complexes with HMG-CoA, CoA, and HMG, and HMG, CoA, and NADP (+) can all be seen in three crystal structures, which provide a clear view of the enzyme's active site. The catalytic parts of human HMGR form tetramers. The crystal structure offers a mechanism for cholesterol sensing and indicates how the oligomeric form of the enzyme affects activity. Since bacterial and human HMGRs have different active site architectures, it could be because of this that bacterial HMGRs have not been found to bind HMGR inhibitors. The produced compound [IV] was more effective than the comparative compound [28]. Table 4. Molecular docking of compound [IV] Docking study C o m p o u n d s B in d in g E n er g y (P L P F it n e ss ) K ca l/ M o l N o . o f A m in o A ci d s In cl u d ed in H -b o n d in g A m in o A ci d s In cl u d ed i n H -b o n d in g n o . o f b o n d in g p o w er o f b o n d in g 1DQ9 66.50 6 ASN 567 1 3.044 ARG 571 3 2.724 2.807 2.927 GLU 719 1 2.863 CYS 561 1 3.044 [IV] 68.00 6 ASN 755 1 2.923 ARG 568 3 3.011 2.745 2.745 GLU 559 1 2.887 SER 865 1 2.838 Figure 1. Molecular docking of 1DQ9 Figure 2. Molecular docking of compound [IV] 3.3 Scanning electron microscope studies (SEM) The SEM was used to approve the morphology and size of polymers and nanocomposites Figure (3) SEM of grafted Chitosan [V], Figure 4 grafted Chitosan blend with PVA [VII], Figure 5 nanocomposites of grafted chitosan /PVA /AgNPs [IX]. The average size of Grafted Chitosan particles [V] ranges between (273-312) nm. While the average size of the particles of Grafted Chitosan blends with PVA[VII] is run between (56-78) nm for the presence of PVA, adding PVA results in the alteration of the blend membrane surface topography and has a considerable impact on the cell spreading. The average nano size of the particles ranges between (31- 48) nm for sliver IHJPAS. 37 (1) 2024 242 NPs; AgNPs have been noticed to have homogenous distributions on the matrix surface. The particles in nanocomposite film were found to have almost spherical morphology. However, some of the accumulations of NPs were also found when the surface was rough [29,30]. Figure 3. The SEM of Grafted Chitosan [V] Figure 4. The SEM of Grafted Chitosan / PVA [VII] Figure 5. The SEM of polymer Nanocomposities [IX] 3.4 Biological activity Grafted chitosan, grafted chitosan blended with PVA, grafted chitosan /PVA with gold or silver nanocomposite tested against two pathogenic bacteria types (G+) Staphylococcus aureus and E. coli (G-) in Table 5, and Figure 6 they all showed excellent inhibition rate, where the nanocomposites were the most activity comparable with Amoxicillin as standard antibiotic. Because of AuNPs' great cell affinity and ease of uptake by immune cells, they can be delivered precisely to the site of infection, where they can inhibit and harm microbial pathogens. The antibacterial effect of silver depends on Ag+, as it binds tightly to electron donor groups in microbial cell walls such as sulfur, nitrogen, or oxygen and enters into the bacterial cell wall, and IHJPAS. 37 (1) 2024 243 the production of free radicals by Ag NPs, which may damage the cell and perforate its membrane [31-35]. Table 5. Antibacterial screening data of some synthesized polymers Comp.No. Escharia .coli Staphylococcus aureus Amoxicillin 17 23 [VI] 23 16 [VIII] 25 16 [XI] 30 29 [XII] 27 15 Figure 6. Antibacterial activities of some polymers and nanocomposites 3.5 Anticancer activity The anticancer activity of various concentrations of some polymers and nanocomposites was investigated against A549 (Human Lung Adenocarcinoma Cells) and REF (R7540) Rat Embryonic Fibroblasts) revealing a good activity, which did not affect the growth of normal Rat Embryonic Fibroblasts. Cell lines were prepared for cytotoxicity assay [36] using cultured cells (96 Wells) in a microtiter plate. The absorbance was measured at (620 nm) on a microplate reader [37]. The cell growth inhibition rate was calculated according to equation [38]: Inhibition rate = 𝑚𝑒𝑎𝑛 𝑜𝑓 𝑐𝑜𝑛𝑡𝑟𝑜𝑙−𝑚𝑒𝑎𝑛 𝑜𝑓 𝑡𝑟𝑒𝑎𝑡𝑚𝑒𝑛𝑡 𝑚𝑒𝑎𝑛 𝑜𝑓 𝑐𝑜𝑛𝑡𝑟𝑜𝑙 × 100 The blend polymer [VIII] and nanocomposites [XI, XII] could selectively permeate cancer cells. All polymer nanocomposites [XI, XII] exhibit good inhibition in concentration (100, 50, 25, 12.5) µg/mL more than the polymer blend [VIII] as in Table 6. Increasing ROS levels and causing damage to the cellular components by intracellular oxidative stress and an increase in glutathione oxidation AgNPs and AuNPs could induce cytotoxicity [39]. Therefore, the anticancer activity of nanocomposites [XI] (AuNPs) showed significant effects at a concentration of 100 μg/mL against the A549 cell line and IC50=16.92 while IC50=82.68 for REF [40], as in Table 7 and Figure7- 11. IHJPAS. 37 (1) 2024 244 Table 6. Inhibition rate of some polymers and naocomposites Table 7. The IC50 of some polymers and naocomposites Treatment IC50 in µg/Ml A549 REF M5 =[VIII] 39.81 72.71 M1=[XI] 16.92 82.68 M3=[XII] 19.41 77.60 Figure7. The IC50 of M5=[VIII](modified chitosan /PVA) Figure 8. The IC50 of M1=[XI] (modified chitosan /PVA+Au) Comp.No. Inhibition rate of 1-g mLμ 12.5-L of Inhibition rate 1-μg mL 25 Inhibition rate of 1-μg mL 50 Inhibition rate of 1-μg mL 100 [VIII] 25.12 54.12 55.11 55.98 [XI] =[VIII] +Au 94.64 94.73 95.13 96.23 [XII]=[VIII] +Ag 86..9 87.2 89.34 90.51 IHJPAS. 37 (1) 2024 245 Figure 9: The IC50 of M3=[XII] (modified chitosan /PVA+Ag) Figure 10. The IC50 of M5 and compare with [M5+Au] and [M5+Ag] A:Image of well for (A549) before Staining B:Image of well for (A549) after Staining C:Image of well for (REF) before Staining D:Image of well for (REF) after Staining Figure 11. A:Image of well for (A549) before Staining, B:Image of well for (A549) after Staining, C:Image of well for (REF) before Staining, and D:Image of well for (REF) after Staining 3.6 Study toxicity test The acute toxicity of some synthesized polymer nanocomposites [XI, XII, XIII) were studied in the Laborator Center for Cancer Research and Medical Genetics according to the Lorke-written method. The study included (25) laboratory mice of the Albino type, with average weights (22-28) gm, three months old, and all males. Mice fasted for 18 hours with free access to water and food before the test. The nanocomposites were dissolved in distilled water and treated through injection. These animals were placed in plastic cages with Metal lids covered with fine sawdust and supplied with water by plastic bottles equipped with food. The treatment group and the control group were compared with doses of the injection, and the study showed after 14 days, no contrast in the weight of the mice daily measured between the group control and the treated groups, and no modification in mice behaviors was carried out, and no toxicity symptoms were reported. Moreover, some mice were sacrificed with cervical dislocation, and kidneys, liver, heart, and lungs were weighed. The visual evaluation of the organs of mice showed normal appearance. These results indicated that polymer nanocomposites have low toxicity towards both investigated organisms [41], as shown on Figure 12 . IHJPAS. 37 (1) 2024 246 A:Image of mice in plastic cages B:Image of mice during injection C: Image of mice through necropsy Figure 12. A:Image of mice in plastic cages, B:Image of mice during injection, and C:Image of mice through necropsy 4. Conclusion The antibacterial activity of synthesized nanocomposites was evaluated in vitro. The results show that the nanocomposites (grafted chitosan/PVA/AuNPs) exhibited very excellent antimicrobial activities comparable with standard antibiotic as Amoxicillin; MTT assay was used to estimate the cytotoxic effect of different concentrations for cancer cell line (A549) of the created nanocomposites and compare with regular cell line (REF), the (grafted chitosan/ PVA/ Au) exhibited very excellent inhibition rate. Finally, a toxicity test for these nanocomposites was studied, and the results showed these nanocomposites were non-toxic. Acknowledgment The authors appreciate the cooperation of the teaching staff in the Department of Chemistry at the College of Education for Pure Science (Ibn Al-Haitham), University of Baghdad. Conflict of Interest The authors declare that they have no conflicts of interest. Funding The research did not receive any financial funding from any institution . 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