Chitosan-CuO Nanoparticles as Antibacterial Shigella dysenteriae : Synthesis , Characterization , and In Vitro Study H )

The synthesis of chitosanCuO nanoparticles was studied. This research’s aims were biosynthesis CuO nanoparticles, synthesis of chitosan-CuO nanoparticles, and used as an antibacterial agent of Shigella dysenteriae.CuO nanoparticles and chitosan-CuO nanoparticles were characterized by FTIR spectroscopy and X-ray diffraction, respectively. CuO nanoparticle was synthesized by the reaction between leaf extract of sweet star fruit (Averrhoa caramhola L.) and copper sulfate pentahydrate. Chitosan-CuO nanoparticles were synthesized by a heating method. The suspension of chitosan-CuO nanoparticles was used as an antibacterial agent with a paper disk method. The result showed that the Cu-0 group at CuO nanoparticles was detected at a wavenumber of 503, 619, 767, and 821 cm 1. The crystallite size of the CuO nanoparticles was 4.25 nm. Cu-0 group bonded at N-H and 0-H groups and detected at 3406 cm 1from the FTIR spectra of chitosan-CuO nanoparticles. The average inhibition zone of chitosan-CuO nanoparticles at concentration 2.500, 5.000, 7.500, and 10.000 ppm to Shigella dysenteriae were 13.57ti.55; 14.90ti.20; 15.97to.76 and 17.03+1.80 mm, respectively. Article history: Received: 5th August 2020 Revised: 11th November 2020 Accepted: 9th January 2021 Online: 31st January 2021


Introduction
Scientists develop metal nanoparticles (MNPs) such as CuO nanoparticles (material scientists, pharmacists, biologists, and chemists). The scientists are developing these MNPs with high-efficiency and low-cost material sources [1]. The CuO nanoparticles have electronic, optical, catalytic, and magnetic activity [2].
Several routes can synthesize CuO nanoparticles. Chemical route (called chemical methods) such as solgel [ 3 ] and precipitation [ 4 , 5 ] and non-chemical route [2]. A non-chemical route is called a green synthesis method. This method is effective, eco-friendly. No need for high temperatures, pressures, and toxic chemicals can be minimized [2]. Toxic chemicals can be substituted by some plant components, such as roots, leaves, stems, seeds, and fruits [6]. Some plant components can act as a bioreduction to produce CuO nanoparticles [1,2,7 ]. The product of CuO nanoparticles can be used as an antibacterial agent [2, 7 , 8], super-strong materials, sensors, and catalysts [2].
Chitosan, an organic polymer, is produced by deacetylation of chitin, and it has the name (1-4 )linked 2-amino 2-deoxy -D glucopyranose [ 9 ]. The properties of chitosan will increase with the modification process at chitosan ' s chemical structure (-NH2 group). The modification process of chitosan can be done by two methods, i.e., physical and chemical methods [10]. Chitosan can be used as supporting materials with metal oxide nanoparticles and applied as antibacterial agents [11,12,13 ]. We studied that chitosan combined with zinc nanoparticles can act as an antibacterial agent. With the increase of concentration of chitosanmetal nanoparticles, its antibacterial agent properties are increasing [10,11]. This fact showed, there is an effect of metal nanoparticles on chitosan. This effect is inhibiting the growth of bacteria. α θ Laha et al. [ 14 ] reported that the synthesis procedure of CuO nanoparticles is easy compared to other metalbased nanoparticles. CuO nanoparticles ' properties are easy to release out of the human body, easily mix with polymers such as chitosan, and relatively stable and unique of chemical and physical properties [ 15 , 16]. CuO nanoparticles combined with chitosan have many advantages, such as the properties of antibacterial [ 13 , 15 , 17 , 18, 19 ]. However, the chitosan modified CuO nanoparticle as antibacterial gram-negative such as Shigella dysenteriae is not widely studied. Shigella dysenteriae is a gram-negative bacterium. The cell wall comprises a thin membrane of peptide polyglycogen, and an outer membrane is lipopolysaccharide phospholipids and lipoprotein [20].
We biosynthesized CuO nanoparticles through a green synthesis. The CuO nanoparticles product can be interacted by chitosan structure through primary amine or OH group of chitosan [ 13 , 21]. FTIR spectroscopy and X-ray diffraction (XRD) are used to characterize chitosan, CuO nanoparticles, and chitosan supported CuO nanoparticles (chitosan-CuO nanoparticles).
Chitosan-CuO nanoparticles were applied as antibacterial of Shigella dysenteriae. Based on the fact that the properties of chitosan as an antibacterial [22] and copper nanoparticles can interact with the bacterial surface [2].

Preparation of aqueous leaf extract of sweet star fruit
About 50 g of sweet star fruit leaves (clean) was used in this study. Cleaned leaves of sweet star fruit were sliced into small shapes and mixed with 100 mL distilled water in a 250 mL Erlenmeyer flask. The mixture was boiled at 90°C ( 15 minutes), and then the mixture could cool at room temperature. The mixture was separated, and the filtrate was stored in the refrigerator for further study [ 23 ].

. Biosynthesis of CuO nanoparticles
Biosynthesis of CuO nanoparticles was synthesized according to Nasrollahzadeh et al. [ 24 ] procedure with slight modification. About 100 mL of leaf extract of sweet star fruit was added to 250 mL Erlenmeyer flask contained 50 mL of 0.1 M CuS 04 5 H2O solution. The mixture was boiled at 8o°C until the mixture ' s color changed from slightly green to deep black. The mixture could cool at room temperature for one night to form precipitation. The residue was separated from filtrate, and residue (CuO nanoparticles) was washed with distilled water (several times). CuO nanoparticles were dried in an electric oven at 50°C until dry.

Synthesis of chitosan supported CuO nanoparticles (chitosan-CuO nanoparticles)
CuO nanoparticles (0.1 g) was suspended in 50 mL of acetic acid 10% (v / v). Chitosan (0.1 g) was added to this solution. The mixture was stirred continuously for 30 min, after which 1 M of KOH solution was added dropwise to the solution until the pH was 10. The solution was continued by heating on a hot plate at 6o°C ( 3 h). Finally, this solution could cool at room temperature for one night to form precipitation. The filtrate was separated from the residue. The residue was washed several times with distilled water until the filtrate of residue has neutral pH. The residue was dried at 50°C in an electric oven until dry ( 3 h) [ 24 ].

. Characterization
The functional group of chitosan-CuO nanoparticles, CuO nanoparticles, and CuS 04 5 H2O were analyzed by FTIR Spectrophotometer (Shimadzu Prestige-21) help of KBr pellets and spectra were recorded at a range of 4500 -500 cm -1 . X-ray diffraction (Shimadzu 6000) was used to calculate the crystalline size of CuO nanoparticles and evaluate the crystalline level of chitosan-CuO nanoparticles, CuO nanoparticles, and copper (II) sulfate pentahydrate. The operational condition of X-ray diffraction is Cu K X-ray tube at 1.5406 A, 30 kV, and 10 mA with scan speed / duration time 10.000 deg. min -1 and the 2 range of o°-6o°.

The preparation of Salmonella Shigella agar as media of the agar diffusion method
The Salmonella Shigella Agar solution was sterilized in an autoclave, 15 mL of Salmonella Shigella Agar was poured into Petri dishes, and then they were solidified as the first layer. 10 mL of fresh inoculum suspension of Shigella dysenteriae (approximately 1.0 x 108 CFU / mL) was spread on each agar plate ' s surface as the second layer.
Finally, the sterile paper disks (6 mm diameter) were dropped by 10 pL the suspension of chitosan-CuO nanoparticles, acetic acid (1% v / v), and chitosan solution, respectively. The paper disk contained the sample was placed aseptically in the second layer. The Petri dishes were incubated at 37°C for 24 h, and the inhibition zones of bacterial growth were measured after 24 h. In vitro study prepared with triplicate.

. Biosynthesis of CuO nanoparticles
The documentation of the biosynthesis of CuO nanoparticles can be seen in Figure 1 acid and alkaline solution [ 29 ]. In the first step, when the acetic acid solution was added to the chitosan compound and CuO nanoparticles, CuO was changed into Cu 2+ ion, and Cu 2+ ion can interact with -OH and -NH2 groups of chitosan structure through the coordination bonds [ 30 ]. In the second step, the addition of alkali compound (OH -) until pH of 10 and the factor of heating, the Cu 2+ ion can be converted again to form CuO [ 17 ]. Schematic reaction and the product chitosan-CuO nanoparticles can be seen in Figures 3 and 4 . I Figure 1. Photograph of water extract of sweet star fruit leaf (a), copper (II) sulfate pentahydrate (b), a mixture of water extract of sweet star fruit leaves and a solution of copper sulfate pentahydrate after boiling at 8o°C (c), the resulting product from CuO nanoparticleswet (d) and dry (e).
The biosynthesis mechanism of CuO nanoparticles is a reduction or an oxidation mechanism [ 24 ]. Sweet star fruit leaf extract contains natural ingredients or metabolites (primary or secondary) such as alkaloids, carbohydrates, glycosides, phytosterols, resins, phenols, tannins, diterpenes, flavonoids, proteins and amino acids, quinones, and phlobatnins [ 25 ]. It had a crucial position in converting metal ions to specific metal nanoparticles. These metabolites are responsible for reducing or oxidizing agents to nano-sized metal oxide [26]. Metal salt, reducing, and stabilizing or capping agents are three components for controlling the size of metal oxide nanoparticles [ 27 ] and illustrated, as seen in   metal oxide nanoparticles with a green synthesis method [28].

. Synthesis of chitosan-CuO nanoparticles
CuO nanoparticles can be bonded by -OH and -NH2 groups of chitosan structure through two steps: effect This fact showed that the chitosan framework is 2, supported by CuO nanoparticles [21], and there is an interaction between the chitosan structure and CuO nanoparticles [ 17 ]. In these spectra, the Cu -0 group ' s stretching vibration appeared at 619 , 721 , and 783 cm 1 [ 7 , 38 , 39 ]. Diffractogram XRD of chitosan-CuO nanoparticles confirmed the evidence of CuO nanoparticles at chitosan structure.

. Analysis of the physical structure
Diffractograms of chitosan, copper (II) sulfate pentahydrate, CuO nanoparticles, and chitosan-CuO nanoparticles were presented in Figure 6. The narrow peaks of copper (II) sulfate pentahydrate in Figure 6a showed an excellent quality crystalline nature and showed the highest peak at 2~180 [ 39 ]. The X-ray diffractogram of chitosan is a semi-crystalline form ( Figure 6b) with two strong diffractions at 2 = io°and 20°. Two strong diffractions showed the semicrystalline and high degree of crystalline morphology of chitosan, respectively. Plenty of 0-H and NH2 groups at the chitosan framework play an important role in intra and intermolecular hydrogen bonds [ 32 ].
The Diffractogram of CuO nanoparticles (Figure 6c) was showed in crystalline. The form of these peaks was sharp and narrow. Diffraction angles of CuO nanoparticles are 15 and were the average crystallite size, the wavelength of X-ray used ( 1.5406 ), the full width at half maximum (FWHM) and the Bragg ' s angle respectively [26]. Figure 6d showed that the diffraction angles of chitosan-CuO nanoparticles are 18.34°, 21.35°, 24.27°, 27.96°, and 56.31°. Chitosan peak at 21.35°a nd another peak of CuO nanoparticles were observed at 56.31° [40 ], which indicates CuO peaks at diffractogram chitosan-CuO nanoparticles although with lesser intensity. The crystallinity of chitosan changed after substituted by CuO nanoparticles. The diffractogram is shown in figure 6d. The effect of decreasing the crystallinity of chitosan, a hydrogen bond in the chitosan structure becomes weak because of the insertion of CuO nanoparticles into the chitosan functional group [ 41 , 42 ]. The prominent bands in the FTIR spectra of chitosan (Figure 5 b) show: the peak at 3427 cm -1 is stretching vibrations of the 0-H group and overlaps with the N-H group. 2920 and 1381 cm 1 are stretching and bending vibration of the C-H group, respectively, and 1656 cm is a bending vibration of the N-H group [ 32 , 33 ]. The deformation vibration of primary amine was detected at 1421 cm -1 [ 34 ]. The peak ati09i cm -1 is stretching vibration of the C-0 group [ 35 ].
FTIR spectra of CuO nanoparticles result from molecular interactions between the media of the aqueous leaf extract of sweet star fruit and CuO nanoparticles (Figure 5 c). The broad peak at 3369 cm 1 is stretching vibration of 0-H groups from alcohols and phenols, but Taranet al. [ 36 ] reported that the band at 3369 cnr 1 is N-H stretching. C-H groups ' stretching vibration appeared at 2848 -2918 cnr 1 [ 7 ]. The peak at 1317 cnr 1 is the C-0 group ' s vibration [ 24 ]. The vibration band at 1099 cm is the stretching vibration of the C = 0 group. C = C stretching detected at 1612 cm 1 . 1444 cnr 1 is a band of 0-H bending, and 1249 cm -1 is C-0 stretching [ 37 ]. The Cu-0 group ' s stretching vibration was detected in wave number 503 , 619 , 767 , and 821 cnr 1 . All these wavenumbers are confirming the formation of CuO nanoparticles [ 7 , 38 , 39 ]. chitosan solution are responsible for the antibacterial activity [18] with the increase of the concentration of chitosan-CuO nanoparticles.
Chitosan-CuO nanoparticles can act as an antibacterial is described by many theories, such as the theory explained by Benhabiles et al. [22]. This theory described an interaction between the positive charge of NH 3 + group chitosan and the negative charge of surface cell bacteria. Interaction of Cu nanoparticles with cell membrane bacteria, and its effect is a decrease of transmembrane electrochemical potential, accumulation on the cell surface, and DNA bacteria causes to destroy cell surface and DNA damage [ 44 ]. Acetic acid has an H + ion, enters the cell cytoplasm of bacteria, and becomes more acidic. The increasing H + ion in the cytoplasm then causes a decrease in the cell ' s local pH and disturbs bacteria ' s growth [ 45 ].   The clear zone of chitosan at a concentration of 2.500 and 5.000 ppm is higher than the chitosan-CuO nanoparticle in the same concentration. Allaker [ 43 ] reported that CuO nanoparticles in a 100-5000 ppm concentration had no toxic effect on cells wall or minimum bactericidal. This fact showed the transparent zone chitosan-CuO nanoparticles is lower than chitosan. On the other hand, the clear zone chitosan-CuO nanoparticles at a 7.500 and 10.000 ppm concentration are higher than chitosan. Chitosan-CuO nanoparticles at a concentration of 7.500 and 10.000 ppm maybe had a toxic effect, and the CuO nanoparticles released from the oxide nanoparticles using Andean blackberry (Rubus glaucus Benth.) fruit and leaf , Journal of Saudi Chemical Society, 21