Document Type : Original Research Article
1 Department of Chemistry, College of Science, University of Anbar, Ramadi, Iraq.
2 Department of Chemistry / College of Education for Pure Science (Ibn Al-Haitham) / University of Baghdad / Iraq
Biopolymers are polymeric biomolecules made up of monomeric components joined together by covalent bonds to form larger molecules. The term "bio" refers to materials that are biodegradable and produced by living creatures . Chitosan a natural polysaccharide is widely utilized in pharmaceuticals, chitosan is produced from chitin and is found as a key component of fungus cell walls, crustacean and insect exoskeletons, and fish scales. It is a cationic polymer made up of (1–4) -2-amino-2-deoxy-glucan that has gotten more attention than chitin because of its pH sensitivity, biocompatibility and bioactive activities . Schiff base derivatives of chitosan are one of the key changes and the newly generated imine groups increase the pharmacological effects of chitosan, such as anticancer, antibacterial and antioxidant activities . Polymer blends are materials created by combining two polymers or copolymers to create a new material with characteristics that complement each other . PVP, also known as povidone or polyvidone is a biocompatible and non-toxic polymer that has been recognized as safe by the Food and Drug Administration. PVP is one of the most promising polymers for nano gel production. Water solubility, toxicity loss, film formation, and adhesive strength are some of its adaptable properties . PVA is a non-toxic, water soluble synthetic polymer with strong physical and chemical characteristics as well as film forming capabilities. PVA is bio inert and it has a variety of applications in medicine, including hemodialysis, nano filtration and composite films. When compared to the characteristics of the individual components, chitosan Schiff base, PVA and PVP give benefits in terms of stability, mechanical strength, and bio compatibility . Nanoparticles have received attention in extensive modern research fields, including electronics, sensors, and medicine. Many different Metal nano particles, such as silver (AgNPs), gold (AuNPs), have been synthesized and investigated in various ways including photochemical synthesis, electrochemical and reduction of solution . Green synthesis aims to avoid producing products that are harmful to the environment through a reliable, sustainable and eco-friendly synthesis process. Therefore, in the green synthesis of metal nanoparticles, microorganisms (e.g. bacteria and fungi) and plant extracts (e.g. root, flower, and fruit) are used as reducing agents, the process for making MNPs from biological materials is easy, economical, environmentally friendly, low-energy, non-toxic and stable . Chitosan nanoparticles have three active hydroxyl groups, a primary and one secondary hydroxy group for each repeated unit, and one amino group for each desacidstylized unit. Chitosan nanoparticles are a hydrophilic polymer with a positive charge. The main factors that affect the adsorption capacity of nano chitosan are its particle size, surface area, molecular weight and degree of deacetylation . Nanocomposites have been utilized in a variety of applications, including wound healing, tissue engineering, thermal treatment and drug delivery. Nanocomposites offer a variety of qualities that make them suitable, including mechanical and thermal capabilities, as well as antibacterial properties . In this work, chitosan nanocomposite with metals (gold and silver) have been prepared, due of its organic application properties, chitosan and chitosan derivatives, particularly those produced through a Schiff base reaction, are the most significant. Recently, the interaction of chitosan with aromatic compounds resulted in the effective synthesis of stable Schiff bases, which are remarkable molecules in many fields of pharmacology and medicine, such as antibacterial and cancer prevention agents . The pH of the environment, the molecular weight of the chitosan and the degree of acetylation of the chitosan have all been shown to influence the amount of chitosan binding to the bacterial cell wall. Low pH in the environment increases the positive charge in the chitosan polymer which facilitates bacterial cell wall binding. . Cancer is a malignant neoplasm illness caused by aberrant and uncontrolled cell development. It is the second leading cause of mortality globally behind heart disease. Its nature is classified as the most hazardous kind of cell proliferation with diverse abnormalities .
Chitosan Schiff base preparation (Cs)
Chitosan (0.5gm) was dissolved in acetic acid 2% and stirred for 1 hour at temperature 60 °C, 4-nitro benzaldehyde (1gm) was dissolved in 30 ml ethanol and stirred for 30 minutes at room temperature and mixing all materials to prepare graft polymer. The mixture was magnetically stirred and heated at a temperature of 60 °C for 18 hours with reflex. After cooling, the crude product was washed with ethanol. The product was dried at 50 °C in oven for 24 hours . The synthetic route of chitosan Schiff base is shown in (Fig. 2).
Polymer Blend Preparation
Polymer blends have been prepared by solution casting method, chitosan Schiff base solution was prepared by dissolution 1 gm of the of Chitosan Schiff base in 2% solution of aqueous acetic acid 50 ml with stirring for 1hour at 60 °C, (5gm) of PVA and PVP have been dissolved in 50 ml of water in order to prepare 10 wt % solutions of the polymer,10 ml Cs, 5 ml PVA and PVP polymer solutions have been mixed to make homogenous solution using hotplate stirrer for 30 minutes. To make the film, the combination solution was cast on Petri-dishes and dried in an oven at 50 °C for 24 hours. Cs / PVA / PVP blends were prepared by mixing different ratio Cs: PVA: PVP (10:5:5) .
Green synthesis of Au and Ag Nano particles
Preparation of crude extract (Onion peels)
Deionized water (100 mL) was added to 10 gm of leaf powder to prepare the onion peels extract. The mixture was stirred and heated at 50 °C for 2 hours, and the resultants were filtered and dried in oven at 50 °C temperature. (0.01gm) of powder product was dissolved in 100 ml deionized water to get onion peels extract (100 ppm). As a stabilizing and reducing agent, fresh extract made from onion peels was utilized. .
Preparation of HAuCl4.3H2O, AgNO3 solutions
Gold chloride trihydrate (HAuCl4.3H2O) (1gm) was dissolved in 100 mL of deionized water to prepare the stock solution, after (2 mL) was taken from stock solution and completed to 100 ml by applying sequential dilution operations to have (100 ppm). AgNO3 was synthesis by dissolving (0.016gm) of AgNO3 in 100 ml deionized water also to have (100 ppm).
Green synthesis of Au and Ag nanoparticles
Aqueous onion peels extract (3 mL) was added to (10 mL) of aqueous gold chloride solution and silver nitrate with sequence, the final mixture was stirrer for 10 minutes at 25 °C . The gold color changed from yellow to purple which confirms the formation of AuNPs, The color of silver was altered from colorless to brown, indicating that AgNPs had formed, The process of separating the nanoparticles is carried out by a centrifuge (10000 rpm) to separate the precipitate from the filtrate, then the precipitate is taken, collected and dilute with deionized water .
Preparation of Cs / PVA / PVP Gold and Silver Nanocomposites
Nanocomposites were prepared by mixing 10 mL of chitosan Schiff base, 5 mL PVA and 5 mL of PVP with 25 mL of two different concentrations (100, 200 ppm) AuNPs and AgNPs with sequence and stirred for 2 hours, the mixture solution was cast onto Petri-dishes and kept in a constant temperature oven at 50 °C in oven for 24 hours to obtain the nanocomposite film . As shown in (Fig. 3).
In vitro bacterial activities polymer blends and Au, Ag nano composites were performed against pathogenic bacteria such as the Acinetobacter baumannii, Staphylococcus aureus, Pseudomonas aeruginosa, and Esherichia coli were collected from Anbar University's Department of Biology, College of Science, where their sensitivity to common antibiotics was also examined by using disk diffusion method. The clear zone of inhibition was measured using Muller-Hinton agar and the agar well diffusion technique. The concentration of polymer blend and nanocomposite were 20 mg. The discs have been placed on seeded plates and incubated for 24 hrs at a temperature of 37 °C. The diameters of inhibition zone which surrounds every disc have been evaluated after (24) hours .
The cells were cultured in RPMI-1640 medium with 10% Fetal bovine serum, 100 units/mL penicillin and 100 g/mL streptomycin. Cells were passaged with Trypsin-EDTA twice a week and reseeded when they reached 80% confluence. [21, 22].
Cancer cell line (AMJ-13) cell line were obtained from the cancer research center in Baghdad. the MTT test was used in 96-well plates to assess the cytotoxic impact of polymer blends and nanocomposites [23, 24]. After 24 hours, or after a confluent monolayer had formed, cells were treated with the tested chemicals at various doses and cell viability was assessed. After 72 hours of treatment, the vitality of the cells was determined by removing the medium, adding 28 liters of a 2 mg/mL MTT solution and incubating the cells for 2.5 hours at 37 °C. After the MTT solution was withdrawn, the crystals in the wells were solubilized by adding 130 µL of DMSO (Dimethyl Sulphoxide) and incubating at 37 °C for 15 minutes with shaking. . The absorbency was measured at 492 nm using a microplate reader and the experiment was carried out three times. The following equation was used to compute the rate of cell growth inhibition (the percentage of cytotoxicity): [26, 27] (Inhibition rate = A- B/A*100), where A is the optical density of the control and B is the optical density of the sample. .
The cells were seeded at a density of 1*105 cells ml-1 into 24-well micro-titration plates and grown for 24 hours at 37 °C to check their shape under an inverted microscope. Polymer blends and Nano composites were exposed to cells at their IC50 concentration for 24 hours. After the exposure time, the plates were stained with crystal violet dye and incubated at 37 °C for 10-15 minutes. The dye was carefully washed out of the stain with tap water until it was completely gone. The cells were shot at a magnification of 100 x using a digital camera attached to an inverted microscope [29, 30, 31].
RESULTS AND DISCUSSION
Characterization of AuNPs and AgNPs.
The preparation AuNPs and AgNPs were characterized by UV-Vis spectroscopy (INOVIALAB-1911DB, China), SEM microscopy (TESCAN Czech Republic), X-ray Powder Diffraction (XRD) (Malvern Panalytical), FTIR (IRAFFINITY-1SHIMHDSO JUPAN), DSC, TGA (Q600).
Spectroscopy in the ultraviolet and visible ranges (UV-Vis)
The UV-Vis spectrum of AuNPs and AgNPs solution is given in (Fig. 4) UV-vis spectroscopy is a method that may validate the production of metal nanoparticles in aqueous solution, (Fig.4) B showed the UV-vis spectra of AgNPs solution, with a broad surface plasmon resonance (SPR) band appearing at 425 nm, indicating the creation of AgNPs. (Fig. 4) A showed the UV-vis spectrum of AuNPs solution with a broad (SPR) band appearing at 543 nm, showing the formation of AuNPs . The (SPR) of AgNPs and AuNPs was used to explain the interaction between free electrons on the metal surface and incoming light. [33, 34].
Scanning Electron Microscopy
The average size and morphology of nanoparticles in the test sample may be determined using SEM imaging, which is an analytical method . (Fig. 5) A,B shows a typical surface of AgNPs and AuNPs prepared from onion peels using green synthesis. The SEM picture revealed a shape of gold Nanoparticles that formed with diameter ranges from (24.74, 57.82, 62.04) nm, the SEM picture revealed a shape of silver nanoparticles that formed with diameter ranges from (18.77, 39.10) nm.
Analysis of X-ray diffraction (XRD)
X-ray diffraction (XRD) analysis was used to assess the crystalline nature of prepared AgNPs and AuNPs. (Fig. 6) A, B and Table 1 are showing the XRD data of produced AuNPs and AgNPs. Both dried AuNPs and AgNPs had a 2theta degree of 10 to 80. Sharp strong peaks were detected at 38°, 44°, 64°, and 77° for AuNPs, which corresponded to the Braggs planes (111), (200), (220), and (311), respectively, according to (JCPDS 04-0784) . confirming that the produced AuNPs possessed a face-centered cubic structure. For AgNPs, the peak obtained at 2theta values 38°, 44°, and 64° correspond to Braggs reflection (111), (200) and (220) respectively, according to (JCPDS 04-0783) .
Characterization of chitosan, chitosan Schiff base and blend
(Fig. 7) A, represents the FT-IR spectrum for chitosan assigned as follows. The broad band at (3267) cm-1 corresponding to (NH and OH stretching vibration), 2920 cm−1 is due to (C-H symmetric stretch), (1621, 1546) cm−1 assigned to amide I, 1369 cm−1 (NH2 bending vibration), 1145 cm−1 (C–O–C bending vibration) and 1061 cm−1 (C–OH stretching vibration), (Fig. 7) B showed a new absorption band at 1647 cm-1 attributed to (C=N) of imine group, The absorption at 1520 cm-1 assigned for the C=C of 4- nitro benzaldehyde, the peak appeared at 810 cm-1 linked to the aromatic range's C-H deformation because stretching vibration of (C–O–C) pairing in β (1-4) glycosidic bonds, additional peaks emerged at 1099 cm-1, For the prepared polymer blend (Fig. 7) C showed a broad band around 3321cm-1 attributed to stretching vibration of hydroxyl group (OH-) of PVA and the secondary amide (–NH)of un reacted chitosan, the band at around 1022 cm-1 indicates the presence of (OH) hydroxyl group with polymeric association and (–NH) a secondary amide, also band appeared at 1427 cm-1 assigned to pyridine ring (C=N) .
Scanning Electron Microscope
Surface morphology, size, crystallinity and phase locations of the produced material may all be studied via SEM examination. . The surface morphology changes for the prepared polymer, chitosan, chitosan Schiff base, polymer blends and polymer nano composite were studied using SEM technique, SEM micrograph for chitosan, chitosan Schiff base / PVA / PVP nano-composite loaded with nano-particles of Au, Ag have been depicted in (Fig. 8)
Thermal Analysis (TGA, DSC)
The Thermo Gravimetric Analysis and Differential Scanning Calorimeter (TGA, DSC) for, chitosan, chitosan Schiff base, chitosan Schiff base / PVA / PVP polymer blend and Chitosan Schiff base / PVA / PVP Au, Ag nanocomposite have been measured in temperature range between 25 °C and 1000 °C with a constant rate which is equal to 10 °C ⁄ min.
TGA curve of chitosan (Fig. 9) A illustrated three stages of a sequence mass lose, the first stage with mass lose (-7.368%) of volatile compounds. The second-step with weight loss approximately (-44.21%) for the side group decomposition, the third stage with weight loss of approximately (-37.46%) for the chain decomposition . DSC curve in the (Fig. 9) A° for chitosan showed a Tg of (85.43 °C). Peak at (317.75 °C) regarding to the polymer melting Tm . TGA curve of chitosan Schiff base (Fig. 9) B illustrated six stages of a sequence mass lose, the first stage with mass lose (-4.396%) of volatile compounds. The second-step with weight loss approximately (-37.99%), the third stage with weight loss of approximately (-6.437%), the forth stage with weight loss of approximately (-13.90%), the fifth stage with weight loss of approximately (-6.27%), the sixth stage with weight loss of approximately (-16.73%) . DSC curve in the (Fig. 9) B° for chitosan Schiff base showed a Tg of (56.12 °C). peak regarding to the crystalline temperature point Tc at (274.72 °C). Peak at (477.76 °C) regarding to the polymer melting Tm .
TGA curve of Chitosan Schiff base / PVA / PVP polymer blend figure (Fig. 9) C illustrated four stages of a sequence mass lose, the first stage with mass lose (-3.529%) of volatile compounds. The second-step with weight loss approximately (-15.53%), the third stage with weight loss of approximately (-63.37%), the forth stage with weight loss of approximately (-9.131%) . DSC curve in the (Fig. 9) C° for Chitosan Schiff base / PVA / PVP polymer blend showed a Tg of (112.12 °C). peak regarding to the Crystalline temperature point Tc at (268.49 °C). Peak at (449.86 °C) regarding to the polymer melting Tm .
The TGA curve of chitosan Schiff base / PVA / PVP-Au nanocomposite (NC-Au) (Fig. 9) D illustrated three stages of a sequence mass lose , the first stage with mass lose (-23.22%) of volatile compounds. The second stage with weight loss approximately (-55.57%), the third stage with weight loss of approximately (-15.67%) for the chain decomposition . DSC curve in the (Fig. 9) D° for NC-Au showed Crystalline temperature point Tc at (293.99 °C). Peak at (445.49 °C) regarding to the polymer melting Tm .
The TGA curve of chitosan Schiff base / PVA / PVP-Ag nanocomposite (NC-Ag) (Fig. 9) E illustrated four stages of a sequence mass lose , the first stage with mass lose (-3.916%) of volatile compounds. The second stage with weight loss approximately (-13.65%), the third stage with weight loss of approximately (-56.44%), the fourth stage with weight loss of approximately (-12.47%) for the chain decomposition . DSC curve in the (Fig. 9) E° for NC-Ag showed a Tg of (125.71 °C), peak regarding to the Crystalline temperature point Tc at (277.19 °C). Peak at (445.36 °C) regarding to the polymer melting Tm .
All temperatures are shifted slightly toward a higher temperature, indicating the influence of the coordination bonding of gold &silver on thermal stability, also it was observed that the blend film show only one Tg as shown on its thermo gram. This indicates the presence of hydrogen bonding interactions between Cs, PVA and PVP in the blend and that these three polymers are well blended together. These findings indicate that the addition of Nano-Au & Ag at such an exceptionally low concentration can improve the thermal stability of Cs/PVA/PVP nanocomposite.
Chitosan is well reported biocompatible and antimicrobial polymer used in various applications. The biological activity of polymer blends, chitosan Schiff base / PVA / PVP /Au, Ag nano composite and antibiotic were tested against four kinds of pathogenic bacteria (Gram +ve and Gram -ve) using Disk Diffusion inhibition method . The results of antibacterial activity and antibiotic were represented in Table 2, 3. The antibacterial properties of chitosan Schiff base, which may be linked to the chemical structure of the studied microorganisms, the presence of the Schiff-base molecule's imine group (C=N) with its 𝜋 - electrons is thought to improve the molecule's lipophilicity making it easier for it to enter the microbe's cell membrane. The antibacterial activity mechanism of nanoparticles differs significantly from that of conventional biocides. The contact of nanoparticles with the cellular membrane is the most essential contribution of nanoparticles in the biocidal process, due to their incredibly tiny size and capacity to permeate it. After that, nanoparticles release reactive species by reacting with the cells' inert environment . Ionic or free radical threats that target enzymes and DNA in the cellular nucleus, causing microbial death or growth inhibition are examples of reactive species . The Inhibition zone of polymer blend and nanocomposites as shown in (Fig. 10). Previous studies, on the other hand, have looked at a variety of ways and substances that can be utilized to inhibit pathogenic bacteria, such as the direct effect of Ag and TiO2 nanoparticles on pathogenic bacteria like Proteus mirabilis and Proteus vulgaris . Using physical forces therapy on dangerous bacteria, such as Audible Sounds and Magnetic Fields, has been shown in several trials to help diminish S. aureus infection resistance .
Anticancer cell line
The cytotoxic effect of blend, Nanocomposites against cancercells was studied. The capacity of the blend, Nanocomposites, to suppress the growth of the breast cancer cell line AMJ-13 was used to assess their anticancer efficacy. The results of this research revealed that blend, nanocomposites had extremely substantial cytotoxic effect against human cancer cell lines. As shown in Fig. 12. The findings show that blends, nanocomposites have the capacity to inhibit the development of cell lines and that this impact is concentration dependent. Inhibition rate and viability of cells of blend and nano composites showed in Table 4. The results showed nanocomposites more inhibitor compared with blend. The results showed cytotoxic effect in AMJ13 cells, IC50 of B = 183.56 µg\ml, IC50 of C =37.78 µg\ml, IC50 of D = 26.04 µg\ml, IC50 of E = 42.61µg\ml, IC50 of F = 35.57 µg\ml . As shown in Fig. 11.
Chitosan Schiff base Cs, Cs / PVA / PVP polymer blend, Cs / PVA / PVP-Au, Ag nanocomposites were prepared. In these synthetic methods, onion peels were used as a reducing and stabilizing agent, UV-vis spectra revealed a characteristic SPR band of 543 nm for AuNPs and 425 nm for AgNPs, and XRD data assessed the crystallinity of these produced nanoparticles. Both AuNPs and AgNPs were face-centered cubic, with average crystallite diameters of 3.96 nm for AuNPs and 3.97 nm for AgNPs for the assign peaks. Addition of AuNPs, AgNPs increasing the biological activity, chitosan Schiff base, polymer blends, Nanocomposites shown activity towards pathogenic bacterial (gram +ve and gram -ve), chitosan Schiff base show more activity than polymer blend, Nanocomposites shown more anticancer activity than polymer blend due existence Au, Ag nanoparticles which increasing activity towards breast cancer cell line AMJ-13.
The authers would like to acknowledge the contribution of the University of Anbar (www. uoanbar.edu.iq) via their prestigious academic staff in supporting this research with all required technical and academic support.
CONFLICTS OF INTEREST
The authors declare that there are no conflicts of interest.