Biocidal Properties of Chitosan-Encapsulated Ternary Titanium Dioxide-Nickel Oxide-Copper Oxide Hybrid Nanomaterials Were Prepared via a Facile One-Pot Precipitation Process

Chitosan-encapsulated ternary titanium dioxide-nickel oxide-copper oxide (CTNC) hybrid nanomaterials (HNM) were synthesised via a facile one-pot precipitation method. The synthesised chitosan-titanium dioxide-nickel oxide-copper oxide was characterised by XRD, UV, FTIR, DLS, FESEM, EDAX, and PL tested against G− (gram-negative) bacterial strains such as K. pneumonia, S. dysenteriae, E. coli, P. vulgaris, P. aeruginosa, and V. cholerae, employed by the well method. The CTNC hybrid nanomaterials exhibit a more substantial antibacterial effect against gram-negative bacteria. The MDA-MB-231 cell-line with an IC50 concentration value of 9.8 g/mL was chosen to test CTNC hybrid nanomaterials’ anticancer properties against human breast cancer cell lines. The toxicity studies of fibroblast L929 cells showed that the CTNC hybrid nanomaterials were less harmful to healthy cells. As a result, the CTNC hybrid nanomaterials can be used for biomedical and industrial applications to improve human health conditions.


Introduction
Cancer is a severe threat to global public health in all parts of the world.Global demographic trends predict that new cancer cases will reach around 20 million annually by 2025 [1].A malignant tumour is a condition where cells ignore the standard functional criteria that regulate cell distribution and grow uncontrollably [2].Chemotherapy is a common and widely used cancer treatment strategy that involves killing rapidly dividing cells, including tumour and normal cells, with severe side effects [3].Furthermore, multidrug-resistant (MDR) bacteria are a significant public health issue.According to the Infectious Diseases Society of America (IDSA), antibiotic resistance is "one of the most serious global health threats" [4].Nanoparticle-based therapy has emerged as an effective treatment option.Because these particles are nanoscale, they have unique physical and chemical properties and can interact with the biological system more efficiently [5].
The multifunctional and adjustable nature of NiO NPs has fascinated the scientific community.NiO NPs are one of the most tailored intrinsic p-type semiconductors with magnetic, electric, electrochemical, catalytic, photocatalytic, and antibacterial properties [6].Even at low concentrations, NiO has a strong antibacterial effect on a wide range of microbes [7].Khashan et al. reported the antibacterial efficacy of NiO NPs against P. aurogenisa, E. coli, S. aureus, and S. pneumonia bacteria, with gram-positive bacteria showing more excellent selectivity [8].Karthik et al. reported that the NiO NPs exhibit higher anticancer properties against the MCF-7 cancer cell line and superior dye degradation for textile blue dye using solar light [9].Faisal et al. demonstrated that CuO, NiO, and Ni/Cu hybrids were very effective against cancer in the HepG2 cell line [10].Bakre et al. (2020) reported that CuO-NiO-TiO2 bimetallic nanocomposites with low metal dopants have better photo-catalytic activity [11].
Chitosan is widely used in the food and bioengineering industries to encapsulate active food ingredients, immobilise enzymes, and serve as a vehicle for controlled medication administration.Chitosan has fascinating properties such as bioavailability, biocompatibility, and bioactivity, in addition to its low toxicity and polycationic nature [12].Pham et al. reported that NiO/chitosan nanocomposites exhibit higher antibacterial effects than gram-negative (Escherichia coli) and gram-positive (Saccharomyces cerevisiae, Bacillus subtilis) bacterial strains [13].However, to the best of our knowledge, the biopolymer (chitosan) with ternary titanium dioxide-nickel oxide-copper oxide derived antibacterial and anticancer properties has not been reported.In this present study, the chitosan-titanium dioxide-nickel oxide-copper oxide (CTNC) hybrid nanomaterial was prepared by using the one-pot chemical method.The resulting CTNC hybrid nanomaterials were characterised for their structural, optical, and biomedical applications, like antibacterial and anticancer activity.

Synthesis of Chitosan-Titanium Dioxide-Nickel Oxide-Copper Oxide (CTNC) Hybrid Nanomaterials
Five hundred-milligramme TiO2 NPs were mixed with 0.1 M nickel(II) nitrate hexahydrate (Ni(NO3)2 6H2O) and 0.1 M copper(II) nitrate hydrate (Cu(NO3)2) salts, and 500 mg chitosan was dissolved in a 50-mL aqueous solution containing 1% acetic acid.The 0.1 M of sodium hydroxide (NaOH) solution was added to the chitosan-TiO2-NiO-CuO solution drop by drop.The brown precipitate has been obtained.The brown precipitate solution was heated at 26 °C for 3 h under a magnetic stirrer.The obtained nanopowder was washed with different deionised water and ethanol solutions.At −3 °C, the nanopowder solution was spun at 15,000 rpm for 40 min.The finished nanopowder was annealed at 800 °C for 5 h.

Characterisation (Supporting Information)
The supporting information described the characterisation and experimental procedure (SI), including the CTNC hybrid nanomaterials (SI1) characterisation.In addition, the testing process investigated the antibacterial activity (SI2), and the MTT assay of CTNC hybrid nanomaterials on the MD-MBA-231 and L929 cell lines (SI3) is given in the SI.

Results and Discussion
The X-ray diffraction pattern of synthesised CTNC hybrid nanomaterials is shown in Fig. 1a.The prepared hybrid nanomaterials exhibit individual binary oxide (TiO2, NiO, and CuO) phases that co-exist in the formation of CTNC hybrid nanomaterials.The single metal oxides TiO2, NiO, and CuO NPs exhibit monoclinic, tetragonal, and cubic structures (Fig. 2).The CuO phase is used for monoclinic crystal structures, TiO2 for tetragonal structures, and NiO for cubic structures.In addition, the typical chitosan diffraction peaks were located at 20.44°.200), (220), and (311), respectively.The average crystallite size of CTNC hybrid nanomaterials was calculated using the Scherrer equation [16].The average crystallite size of CTNC hybrid nanomaterials was calculated at 62 nm.The UV-visible spectroscopy analysis of CTNC hybrid nanomaterials' specific surface plasmon resonance (SPR) peaks at 397 nm (Fig. 1b) [17].Figure 1c shows the FTIR spectra of CTNC hybrid nanomaterials.The characteristic absorption bands of chitosan are situated at 3427, 2922, 2845, 1642, 1565, and 1024 cm −1 , corresponding to the-OH and-NH with a hydroxyl group, the C-H asymmetric, the C-H symmetric, and the amide group from the C=O stretching, the -NH2 scissoring vibration, and the C=O stretching (Cao et al. 2016; Duan et al. 2015a) [18].In the FTIR spectra of the synthesised CTNC hybrid nanomaterials observed at 471 cm −1 , the typical metal-oxygen absorption bonds were confirmed [19][20][21].In chitosan (Fig. 3), several characteristic peaks were observed.The broad peaks at 3448 cm −1 and 1637 cm −1 correspond to the stretching vibrations of the hydroxyl (-OH) and amino (-NH) groups, respectively.These peaks indicate the presence of hydrogen bonding within the chitosan structure.Another significant rise at 1381 cm −1 is attributed to the COO-group, corresponding to the carboxylic acid salt within the chitosan molecule.This peak provides evidence of carboxylate groups' presence in the chitosan structure.These peaks confirmed that chitosan molecules successfully interacted with copper-titanium and nickel of the CTNC hybrid nanomaterials and that these interactions were attributed to the electrostatic interaction between chitosan-titanium-nickel-copper oxide surface matrices (Fig. 4).
As shown in Fig. 1d, DLS analysis was used to estimate the average size of the prepared CTNC hybrid nanomaterials.
The hydrodynamic diameter was 207 nm for CTNC hybrid nanomaterials.FESEM images (Fig. 1e) show that the CTNC hybrid nanomaterials exhibit trigonal-structure nanospheres, and the average particle size is 50-60 nm (Fig. 5).In the case of NiO, CuO and TiO NPs exhibit a polygonal structure with an agglomerated form.Also, it was observed that the surfaces of the spheres were not smooth.It was made up of many small nanoparticles.In addition, particles formed randomly oriented The UV emission (NBE emission: near band edge emission), observed 365 and 394 nm, corresponds to the recombination between electrons in the conduction band and holes in the valence band.The band edge-free excitons are responsible for the visible emission peaks at 418, 448, 482, and 523 nm [22].In photo-catalysts, the excitonic PL signal in the 400-550-nm range is caused by flaws or surface oxygen vacancies [23].The antibacterial activity of CTNC hybrid nanomaterials was tested using the well diffusion method against gram-negative bacteria such as K. pneumonia, S. dysenteriae, E. coli, P. vulgaris, P. aeruginosa, and V. cholerae, with concentrations of 1-2 mg/mL shown in Fig. 6b.CTNC hybrid nanomaterials and the conventional antibiotic amoxicillin show antibacterial effects.CTNC exhibits more antibacterial activity as compared to amoxicillin.Increasing the concentration of the hybrid nanomaterials also increased the antibacterial activity.The antibacterial mechanisms for the CTNC hybrid nanomaterials' interaction with bacteria have been explained.The bacterial cell membrane surface attaches to CTNC hybrid nanomaterials, inhibiting protein synthesis and nutrient intake and disrupting cell growth and viability.On the other hand, the generation of reactive oxygen species (ROS) can interact with bacterial membranes, causing cell walls to lyse and eventually die [24].
The cytotoxicity of the CTNC hybrid nanomaterials was tested at different concentrations from 2.5 to 15 μg/mL.As shown in Fig. 7a1-a3, the IC 50 value of 9.8 μg/mL was p≤0.05 after 24 h of CTNC nanomaterial treatment with MDA-MB-231 cells.The particle size of the CTNC hybrid nanomaterials was observed at ∼48.37 nm through XRD, and oxygen vacancies (O v ) were observed at 523 nm, as determined by PL studies.These defects increase the number of electron-hole pairs that can migrate onto the surface of the CTNC hybrid nanomaterial matrix.In comparison, the electrons and holes can react with the active free radicals such as OH − , H 2 O 2 , and 1 O 2 , CTNC hybrid nanomaterials in their aqueous environment, which is responsible for the generation of ROS.These results show cells can oxidise and damage DNA, lipids, and proteins in the cellular environment [24].
The toxicity of nanoparticles depends on several parameters, such as protein adsorption, dissolution rate, reactive oxygen species (ROS), and the release of metal Cu, Ni, and Ti ions.An optical microscope was used to employ the L929 fibroblast cells, as shown in Fig. 7b1-b3.The control cells showed 100% viable cells and a uniform fusiform structure.In addition, the prepared CTNC HNM-treated L929 fibroblast cells were alive, with a viability of 82.53% at the maximum dose, i.e. 15 μg/mL, indicating that CTNC hybrid nanomaterials have low toxicity.The CTNC nanomaterial, on the other hand, may be used as a non-toxic supplement in cosmetic products [24].

Conclusions
In summer, the chitosan encapsulates titanium dioxide-nickel oxide-copper oxide HNM synthesised via a facile one-pot precipitate method.The XRD patterns reveal that the synthesised CTNC hybrid nanomaterials exhibit individual binary oxide (TiO2 (anatase), NiO (cubic), and CuO (monoclinic)) phases that co-exist in the formation of CTNC hybrid nanomaterials.The typical metal-oxygen absorption bonds were confirmed from the FTIR spectra of the synthesised CTNC hybrid nanomaterials observed at 772, 584, 525, and 471 cm −1 .The FESEM image of synthesised CTNC hybrid nanomaterials reveals trigonal-structure nanospheres, and the average particle size is 50-60 nm.The CTNC hybrid nanomaterials and the conventional antibiotic amoxicillin show antibacterial effects.CTNC exhibits more antibacterial activity as compared to amoxicillin.Increasing the concentration of the hybrid nanomaterials also increased the antibacterial activity.The CTNC hybrid nanomaterials were tested using an MTT assay on a breast cancer cell line (MDA-MB-231 cells), and the results demonstrated that CTNC hybrid nanomaterials have more anti-cancer properties.Toxicity tests on fibroblast L929 cells revealed that CTNC hybrid nanomaterials were less toxic to healthy cells.