# CuO-TiO2-Chitosan-Berbamine Nanocomposites Induce Apoptosis through the Mitochondrial Pathway with the Expression of P53, BAX, and BCL-2 in the Human K562 Cancer Cell Line

Authors: Abozer Y. Elderdery; Badr Alzahrani; Siddiqa M. A Hamza; Gomaa Mostafa-Hedeab; Pooi Ling Mok; Suresh Kumar Subbiah
Journal: Bioinorganic Chemistry and Applications
 (2022)

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## Abstract

In this study, cells from human Chronic Myelogenous Leukemia (K562) were cultivated with CuO-TiO2-Chitosan-Berbamine nanocomposites. We examined nanocomposites using XRD, DLS, FESEM, TEM, PL, EDAX, and FTIR spectroscopy, as well as MTT for cytotoxicity, and AO/EtBr for apoptotic morphology assessment. The rate of apoptosis and cell cycle arrests was determined using flow cytometry. Flow cytometry was also employed to identify pro- and antiapoptotic proteins such as Bcl2, Bad, Bax, P53, and Cyt C. The FTIR spectrum revealed that the CuO-TiO2-Chitosan-Berbamine nanocomposites were electrostatically interlocked. The nanocomposites’ XRD signals revealed a hexagonal shape. In the DLS spectrum, nanocomposites were found to have a hydrodynamic diameter. As a result of their cytotoxic action, nanocomposites displayed concentration-dependent cytotoxicity. The nanocomposites, like Doxorubicin, caused cell cycle phase arrest in K562 cells. After treatment with IC50 concentrations of CuO-TiO2-Chitosan-Berbamine nanocomposites and Doxorubicin, a substantial percentage of cells were in G2/M stage arrest. Caspase-3, -7, -8, -9, Bax, Bad, Cyt C, and P53 expression were considerably enhanced in K562 cells, whereas Bcl2 expression was decreased, indicating that these cells may have therapeutic potential against human blood cancer/leukemia-derived disorders. As a result, the nanocomposites demonstrated outstanding anticancer potential against leukemic cells. CuO-TiO2-Chitosan-Berbamine, according to our findings.


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## Body

## 1. Introduction

Cancer of white blood cells is known as chronic myeloid leukemia (CML), resulting from the reciprocal translocation of the t (9; 22) gene in the bone marrow [1]. CML is characterized by the unregulated growth of myeloid cells in the bone marrow and an accumulation of these cells in the bloodstream. Patients with chronic myeloid leukemia are found to have the BCR-ABL1 oncokinase [2]. A mainstay of treatment in CML is tyrosine kinase inhibitors (TKIs) such as imatinib, bosutinib, dasatinib, and nilotinib, which can be used as first-line chemotherapy [3]. CML is only caused by high levels of radiation exposure, such as in the case of a nuclear reactor accident or a survivor of an atomic bomb blast. Age and gender are the two most important risk factors for CML [4]. CML becomes more frequent as you become older, and for unknown reasons, males are significantly more likely to get it than females. Smoking, nutrition, chemical exposure, or infections appear to have little effect on the incidence of CML [5].The American Cancer Society predicts that around 8,860 (5120 men and 3740 women) new cases of CML will be detected in the United States in 2022. Approximately 1,220 people will die as a result of CML (670 men and 550 women). Chronic myeloid leukemia accounts for around 15% of all new leukemia cases [6]. One in every 526 people in the United States will acquire CML during their lifetime. CML is diagnosed at the age of 64 on average. Nearly half of all instances are reported by those aged 65 and above [7]. This kind of leukemia primarily affects adults, while children are rarely afflicted. This form of blood disorder is being studied in laboratories and clinical trials around the world [8].TKI medicines are being explored in clinical trials to evaluate if greater doses or combinations with other treatments, such as chemotherapy or interferon, are better than either one alone [9]. CML is a kind of leukemia caused by BCR-ABL gene alterations. TKI resistance can develop as a result of mutations [10]. Other medications, such as immunological and chemotherapeutic agents, are also undergoing clinical studies. Several vaccinations are being developed for the treatment of CML [11, 12].The development of hybrid composites has increased in recent years due to demonstration of high bactericidal activities, making them suitable for wide range of applications including water treatment [13]. Different techniques can be used to create hybrid nanocomposites, including organic-organic and organic-inorganic processes, as well as inorganic-inorganic processes such as TiO2-ZnO, TiO2, and TiO2-MgO [14]. Thin films, superlattices, nanograins, polymer intercalation, sol-gel synthesis, thermal deposition, chemical and physical vapor deposition, suspension, and liquid phase deposition are currently accessible coating technologies for thermoelectric metal oxides [15].These approaches are useful in improving each component’s technological and mechanical properties while also revealing new functionalities. Inorganic materials like titanium dioxide (TiO2) are currently being used in combination with natural polymers like chitosan (CS) to create composites (CS-TiO2) with beneficial properties [16, 17]. Chitosan (CS) is a naturally occurring biopolymer that is a linear polysaccharide containing amino-deoxy-D-glucans that are linked with 1–4 linkage that is formed by deacetylating chitin, the major structural component of crustacean exoskeletons [18]. CS is nontoxic and biodegradable, with a polycationic nature. CS is a physiologically active molecule that possesses several fascinating features. It can be used to create edible coatings, packaging, and drug-eluting carriers for food and medicinal applications [16, 19].Titanium dioxide (TiO2) is a versatile and chemically inert substance used in a wide range of applications such as food, medicines, biomedical, antibacterial agents, and environmental applications [20]. TiO2’s physicochemical, mechanical, photocatalytic, and thermal qualities, as well as its low price, safe manufacture, and biocompatibility, contributeto its extensive application. When coupled with biopolymers such as chitosan gums and starch, nano-TiO2 has been found to minimize the spontaneous agglomeration of TiO2.Berbamine is a bis-benzylisoquinoline alkaloid produced from berberis that is used in China to treat leukopenia and operates as calmodulin antagonist properties [21, 22]. Berbamine is a natural alkaloid with numerous pharmacological effects, including antibacterial and anticancer activities. In hepatoma, leukemia, and colon cancer cells, berbamine can activate caspases and induce apoptosis [23–26]. Berbamine has also been proven to decrease lung cancer growth and migration, as well as blocking metastatic breast cancer cell development [27, 28]. ZnO and CuO nanoparticles are largely utilized in cancer therapy and cosmetics, as well as in industrial operations as catalysts. Zinc oxide nanoparticles (NPs) are commonly used because they generate reactive oxygen species and have a distinct electrostatic characteristic that can help avoid DNA damage [29]. The goal of this study was to show a straightforward way of creating hybrid nanocomposites by combining titanium oxide (TiO2) and copper oxide (CuO) with the natural polymer chitosan and the plant-derived alkaloid berbamine.These nanocomposites are commonly employed as nano delivery systems that improve bioavailability, biodistribution, and preferentially localizing diseased tissues while protecting healthy tissues. However, because of their chemical nature, many metal ion (TiO2/CuO) based nanoparticles also demonstrate effective antibacterial activity [30, 31]. The antibacterial potential of a Berberis vulgaris plant extract against microorganisms linked with caries, such as Streptococcus spp. and Lactobacillus rhamnosus, was determined in several in vitro studies [32]. These nanoparticles and phytocompounds are powerful antibacterial and anticancer agents on their own; however, combining them with the natural polymer chitosan could result in a new hybrid treatment strategy that delivers better results and overcomes individual flaws. Consequently, the goal of this research is to evaluate if ZnO-TiO2-Chitosan-Berbamine nanocomposites have a synergistic anticancer effect against human CML (K562 cells).


## 2. Material Methods

### 2.1. Synthesis of CuO-TiO2-Chitosan-Berbamine Nanocomposites (CTCBNc)

0.5 g of TiO2 NPs were mixed with 0.1 M Cu (NO3)2.6H2O in 50 mL of the aqueous solution, and 0.5 g of chitosan was dissolved in 1% acetic acid in 50 mL of the aqueous solution. The CuO-TiO2-Chitosan solution was also mixed with 50 mg of berbamine, a phyto component. To create the black residue, 0.1 M NaOH solution was added drop by drop to the CuO-TiO2-Chitosan-Berbamine solution. The residue was boiled at room temperature for 3 hours using a magnetic stirrer. Various stages of nanopowder generation were washed with deionized sterile water and ethyl alcohol solutions. The solution was centrifuged at 15,000 rpm for 40 minutes at −3°C. The black residue was dried for 2 hours at 120°C before being calcined for 5 hours at 600°C.


### 2.2. Spectral Analysis of CTCBNc

CuO-TiO2-Chitosan-Berbamine samples were characterized using an X-ray diffractometer (XRD) (model: X'PERT PRO PANAlytical) using a monochromatic CuK diffraction beam with a wavelength of 1.5406. The CuO-TiO2-Chitosan-Berbamine system was examined using an Energy Dispersive X-ray Spectrometer (EDX) (model: Inca) and a field emission scanning electron microscope (Carl Zeiss Ultra 55 FESEM). We investigated the morphologies of CuO-TiO2-Chitosan-Berbamine using a TEM (Tecnai F20 model) microscope and a 200 kV accelerating voltage. FTIR spectra were collected with a Perkin-Elmer spectrometer in the 400–4000 cm-1 range, absorption spectra of nanocomposites with a Lambda spectrometer in the 200–1100 nm range, and photoluminescence (PL) spectra with a PerkinElmer-LS.


### 2.3. Antimicrobial Activity of CTCBNc

Through the good diffusion method, antibacterial activity targets microorganisms, includingBacillus subtilis, Streptococcus pneumonia, Klebsiella pneumonia, and Proteus vulgaris bacterial strains. To investigate the antibacterial performance of CuO-TiO2-Chitosan-Berbamine nanocomposites dispersed in a 5% sterilized dimethyl sulphoxide solution, 25 mL of Mueller Hinton agar media containing sterile Petri plates was streaked with bacterial pathogens. Positive control was carried out by using amoxicillin (30 µg). The experiments were performed in triplicate on Petri plates incubated overnight at 37°C for 24 hours, and the zones' sizes were measured.The antifungal properties of CTCBNc onCandida albicans were determined by the agar well diffusion method and growth on potato dextrose agar (PDA). The C. albicans strain can be inoculated onto the PDA agar Petri plate by streaking 2–3 times with uniform distribution of inoculum. Following that, sterile forceps were used to place wells containing 1, 1.5, and 2 mg/mL of CTCBNc onto the inoculated plates, which were then incubated for 24 hours at 30°C under visible light. The zone was measured, and the assays were run in triplicate with amoxicillin (30 µg) as a positive control.


### 2.4. Materials for Cell Culture

Sigma Chemical Company provided Dulbecco's Modified Eagle Medium (DMEM), Ethidium bromide (EtBr), Acridine orange (AO), and fetal bovine serum (FBS) (Germany). Biowest France supplied the penicillin-streptomycin. SPL furnished the culture plates (Korea). Sigma Aldrich provided dimethyl sulphoxide (DMSO) and MTT [3-(4, 5-dimethyltiazol-2-yl)-2, 5 diphenyltetrazolium bromide] (Germany). Other materials included propidium iodide (PI), Caspase-3 FITC antibody (BD Biosciences, USA), Caspase-7 FITC antibody (Abbexa Ltd, UK), Caspase-8 PE antibody (Abcam, USA), Caspase-9 FITC staining kit (Abcam, USA), Anti-Bad FITC antibody (BD Biosciences, CA, USA), Purified Bax antibody (Biolegend, USA) (BD Biosciences, USA).


### 2.5. Culture of Chronic Myelogenous Leukemia (K562) Cell Line

The K562-human blood cancer cells were cultured in DMEM, which was supplemented with 10% FBS, penicillin, and streptomycin. To establish confluency, the K562 cells were incubated at 37°C in a humidified environment with 5% CO2.


### 2.6. A Study of the Cytotoxicity of CTCBNc

To assess nanocomposites that are cytotoxic to cells, an MTT-based cell viability experiment was performed. The adhesion of K562 cells to the 96 well microtiter plates. In triplicate, 100 mL of fresh culture media containing 6.25, 12.5, 25, 50, 100, and 200µg/mL of CuO-TiO2-Chitosan-Berbamine were added, followed by a 24-hour incubation at 37°C in a 5% CO2 incubator with the untreated and positive control (Doxorubicin). A 100 µL of MTT (5 mg/mL) was added to the cells for 4–5 hours, followed by a 15-minute addition of DMSO solution to dissolve the bottom layer of crystals. This was followed by the optical density (OD) measurement of the formazan product and calculation of the IC50 [33].


### 2.7. Method for Determining Apoptosis in Cells Using AO/EtBr

5X104 cells/mL in culture medium were treated for 24 hours with CuO-TiO2-Chitosan-Berbamine nanocomposites at the IC50 concentration. Then, the acridine orange/ethidium bromide (AO/EtBr) staining was used to detect apoptotic cells. An inverted microscope was used to detect the cell morphology after this time (Olympus, Germany). The cells were also stained with 1 µL of AO/EtBr solution (100 mg/mL) before being examined under a fluorescence microscope (Olympus BX41, Germany).


### 2.8. CuO-TiO2-Chitosan-Berbamine Nanocomposites Causes an Arrest in Cell Cycle

In this method, propidium iodide (PI) was used to stain the DNA, and the cells were divided into four main phases of the cell cycle (sub-G1, G1, S, and G2/M) according to the DNA content of their cells. We treated K562 cells with IC50 concentrations of CTCBNc nanocomposites for 24 hours, fixed them in 70% ethanol, and washed them twice in 1 X PBS at −20°C after treatment. After staining with propidium iodide at 50 g/mL, the cells were incubated at 37°C for 10 minutes in a dark room. As a result of flow cytometry, the percentage of cells in each phase was calculated using CellQuest Software.


### 2.9. Apoptosis Pathway Detected by Activation of Caspase-3, 7, 8, and 9

According to instructions provided by the manufacturer, caspase-3, 7, 8, and 9 activation levels were measured using FITC/PE antibodies supplied by BD Pharmingen (San Diego, CA, USA). The cells were incubated at 37°C for 24 hours in an incubator at a density of 2 × 105cells/2 mL in a 6-well plate. Then, the cells were added with the nanocompounds at IC50 concentration. The cells were then washed with 1 X PBS before harvesting with 200 µL of the trypsin-EDTA solution. The trypsinization was stopped by adding 2 mL of the medium before incubation for 3–4 minutes. The cells in the tubes were then concentrated for five minutes at 300 × g at 25°C, decanted into PBS, and then dissolved in 1 mL 70 percent ethanol (ice cold). To stain the cells with the desired antibody, the cells were incubated in 500 µL of PBS saline , and mixed well, before being incubated for 15–30 minutes in the dark with 20 µL of the desired antigen (Caspases-3, 7, 8, and 9). CellQuest software was used to generate histograms for samples analyzed on a FACS Calibur Flow Cytometer (Becton Dickinson, San Jose, CA, USA), using channels FL1 and FL2 for FITC and PE-conjugated antibodies, respectively.


### 2.10. Quantification of Bcl2, Bad, Bax, P53, and Cyt C Expression by Flow Cytometry

A FITC/PE antibody obtained from BD Pharmingen (San Diego, CA, USA) was used to measure the activation of Bcl2, Bad, Bax, P53, and Cyt C. Following treatment with IC50 concentration of compounds, the cells were trypsinized with 200 µL of the trypsin-EDTA solution, and then added with 2 mL of the medium to stop the reaction. The cells in the tubes were concentrated for five minutes at 300 × g at 25°C, decanted into PBS, and then dissolved in 1 mL 70 percent ethanol (ice cold). To stain the cells with the desired antibody, the cells were incubated in 500 µL of PBS saline , and mixed well, before being incubated for 15–30 minutes in the dark with 20 µL of the desired antigen (Bcl2, Bad, Bax, P53, and Cyt C). CellQuest software was used to generate histograms for samples analyzed on a FACS Calibur Flow Cytometer (Becton Dickinson, San Jose, CA, USA), using channels FL1 and FL2 for FITC and PE-conjugated antibodies, respectively.


### 2.11. Statistical Analysis

SPSS software version 24 was used to analyze the data. The IC50 was determined using GraphPad Prism software V8 and linear regression analysis (version 8, USA). The experiments were carried out three times, and the mean and standard deviation were computed. The Student’s t-test was used to compare groups (two-tailed), and statistical significance was established as a P<0.05.


## 2.1. Synthesis of CuO-TiO2-Chitosan-Berbamine Nanocomposites (CTCBNc)

0.5 g of TiO2 NPs were mixed with 0.1 M Cu (NO3)2.6H2O in 50 mL of the aqueous solution, and 0.5 g of chitosan was dissolved in 1% acetic acid in 50 mL of the aqueous solution. The CuO-TiO2-Chitosan solution was also mixed with 50 mg of berbamine, a phyto component. To create the black residue, 0.1 M NaOH solution was added drop by drop to the CuO-TiO2-Chitosan-Berbamine solution. The residue was boiled at room temperature for 3 hours using a magnetic stirrer. Various stages of nanopowder generation were washed with deionized sterile water and ethyl alcohol solutions. The solution was centrifuged at 15,000 rpm for 40 minutes at −3°C. The black residue was dried for 2 hours at 120°C before being calcined for 5 hours at 600°C.


## 2.2. Spectral Analysis of CTCBNc

CuO-TiO2-Chitosan-Berbamine samples were characterized using an X-ray diffractometer (XRD) (model: X'PERT PRO PANAlytical) using a monochromatic CuK diffraction beam with a wavelength of 1.5406. The CuO-TiO2-Chitosan-Berbamine system was examined using an Energy Dispersive X-ray Spectrometer (EDX) (model: Inca) and a field emission scanning electron microscope (Carl Zeiss Ultra 55 FESEM). We investigated the morphologies of CuO-TiO2-Chitosan-Berbamine using a TEM (Tecnai F20 model) microscope and a 200 kV accelerating voltage. FTIR spectra were collected with a Perkin-Elmer spectrometer in the 400–4000 cm-1 range, absorption spectra of nanocomposites with a Lambda spectrometer in the 200–1100 nm range, and photoluminescence (PL) spectra with a PerkinElmer-LS.


## 2.3. Antimicrobial Activity of CTCBNc

Through the good diffusion method, antibacterial activity targets microorganisms, includingBacillus subtilis, Streptococcus pneumonia, Klebsiella pneumonia, and Proteus vulgaris bacterial strains. To investigate the antibacterial performance of CuO-TiO2-Chitosan-Berbamine nanocomposites dispersed in a 5% sterilized dimethyl sulphoxide solution, 25 mL of Mueller Hinton agar media containing sterile Petri plates was streaked with bacterial pathogens. Positive control was carried out by using amoxicillin (30 µg). The experiments were performed in triplicate on Petri plates incubated overnight at 37°C for 24 hours, and the zones' sizes were measured.The antifungal properties of CTCBNc onCandida albicans were determined by the agar well diffusion method and growth on potato dextrose agar (PDA). The C. albicans strain can be inoculated onto the PDA agar Petri plate by streaking 2–3 times with uniform distribution of inoculum. Following that, sterile forceps were used to place wells containing 1, 1.5, and 2 mg/mL of CTCBNc onto the inoculated plates, which were then incubated for 24 hours at 30°C under visible light. The zone was measured, and the assays were run in triplicate with amoxicillin (30 µg) as a positive control.


## 2.4. Materials for Cell Culture

Sigma Chemical Company provided Dulbecco's Modified Eagle Medium (DMEM), Ethidium bromide (EtBr), Acridine orange (AO), and fetal bovine serum (FBS) (Germany). Biowest France supplied the penicillin-streptomycin. SPL furnished the culture plates (Korea). Sigma Aldrich provided dimethyl sulphoxide (DMSO) and MTT [3-(4, 5-dimethyltiazol-2-yl)-2, 5 diphenyltetrazolium bromide] (Germany). Other materials included propidium iodide (PI), Caspase-3 FITC antibody (BD Biosciences, USA), Caspase-7 FITC antibody (Abbexa Ltd, UK), Caspase-8 PE antibody (Abcam, USA), Caspase-9 FITC staining kit (Abcam, USA), Anti-Bad FITC antibody (BD Biosciences, CA, USA), Purified Bax antibody (Biolegend, USA) (BD Biosciences, USA).


## 2.5. Culture of Chronic Myelogenous Leukemia (K562) Cell Line

The K562-human blood cancer cells were cultured in DMEM, which was supplemented with 10% FBS, penicillin, and streptomycin. To establish confluency, the K562 cells were incubated at 37°C in a humidified environment with 5% CO2.


## 2.6. A Study of the Cytotoxicity of CTCBNc

To assess nanocomposites that are cytotoxic to cells, an MTT-based cell viability experiment was performed. The adhesion of K562 cells to the 96 well microtiter plates. In triplicate, 100 mL of fresh culture media containing 6.25, 12.5, 25, 50, 100, and 200µg/mL of CuO-TiO2-Chitosan-Berbamine were added, followed by a 24-hour incubation at 37°C in a 5% CO2 incubator with the untreated and positive control (Doxorubicin). A 100 µL of MTT (5 mg/mL) was added to the cells for 4–5 hours, followed by a 15-minute addition of DMSO solution to dissolve the bottom layer of crystals. This was followed by the optical density (OD) measurement of the formazan product and calculation of the IC50 [33].


## 2.7. Method for Determining Apoptosis in Cells Using AO/EtBr

5X104 cells/mL in culture medium were treated for 24 hours with CuO-TiO2-Chitosan-Berbamine nanocomposites at the IC50 concentration. Then, the acridine orange/ethidium bromide (AO/EtBr) staining was used to detect apoptotic cells. An inverted microscope was used to detect the cell morphology after this time (Olympus, Germany). The cells were also stained with 1 µL of AO/EtBr solution (100 mg/mL) before being examined under a fluorescence microscope (Olympus BX41, Germany).


## 2.8. CuO-TiO2-Chitosan-Berbamine Nanocomposites Causes an Arrest in Cell Cycle

In this method, propidium iodide (PI) was used to stain the DNA, and the cells were divided into four main phases of the cell cycle (sub-G1, G1, S, and G2/M) according to the DNA content of their cells. We treated K562 cells with IC50 concentrations of CTCBNc nanocomposites for 24 hours, fixed them in 70% ethanol, and washed them twice in 1 X PBS at −20°C after treatment. After staining with propidium iodide at 50 g/mL, the cells were incubated at 37°C for 10 minutes in a dark room. As a result of flow cytometry, the percentage of cells in each phase was calculated using CellQuest Software.


## 2.9. Apoptosis Pathway Detected by Activation of Caspase-3, 7, 8, and 9

According to instructions provided by the manufacturer, caspase-3, 7, 8, and 9 activation levels were measured using FITC/PE antibodies supplied by BD Pharmingen (San Diego, CA, USA). The cells were incubated at 37°C for 24 hours in an incubator at a density of 2 × 105cells/2 mL in a 6-well plate. Then, the cells were added with the nanocompounds at IC50 concentration. The cells were then washed with 1 X PBS before harvesting with 200 µL of the trypsin-EDTA solution. The trypsinization was stopped by adding 2 mL of the medium before incubation for 3–4 minutes. The cells in the tubes were then concentrated for five minutes at 300 × g at 25°C, decanted into PBS, and then dissolved in 1 mL 70 percent ethanol (ice cold). To stain the cells with the desired antibody, the cells were incubated in 500 µL of PBS saline , and mixed well, before being incubated for 15–30 minutes in the dark with 20 µL of the desired antigen (Caspases-3, 7, 8, and 9). CellQuest software was used to generate histograms for samples analyzed on a FACS Calibur Flow Cytometer (Becton Dickinson, San Jose, CA, USA), using channels FL1 and FL2 for FITC and PE-conjugated antibodies, respectively.


## 2.10. Quantification of Bcl2, Bad, Bax, P53, and Cyt C Expression by Flow Cytometry

A FITC/PE antibody obtained from BD Pharmingen (San Diego, CA, USA) was used to measure the activation of Bcl2, Bad, Bax, P53, and Cyt C. Following treatment with IC50 concentration of compounds, the cells were trypsinized with 200 µL of the trypsin-EDTA solution, and then added with 2 mL of the medium to stop the reaction. The cells in the tubes were concentrated for five minutes at 300 × g at 25°C, decanted into PBS, and then dissolved in 1 mL 70 percent ethanol (ice cold). To stain the cells with the desired antibody, the cells were incubated in 500 µL of PBS saline , and mixed well, before being incubated for 15–30 minutes in the dark with 20 µL of the desired antigen (Bcl2, Bad, Bax, P53, and Cyt C). CellQuest software was used to generate histograms for samples analyzed on a FACS Calibur Flow Cytometer (Becton Dickinson, San Jose, CA, USA), using channels FL1 and FL2 for FITC and PE-conjugated antibodies, respectively.


## 2.11. Statistical Analysis

SPSS software version 24 was used to analyze the data. The IC50 was determined using GraphPad Prism software V8 and linear regression analysis (version 8, USA). The experiments were carried out three times, and the mean and standard deviation were computed. The Student’s t-test was used to compare groups (two-tailed), and statistical significance was established as a P<0.05.


## 3. Results

### 3.1. Spectral Characteristics of CuO-TiO2-Chitosan-Berbamine Nanocomposites in the UV-Visible Range

The UV-Visible absorbance spectrum of the nanocomposites CuO-TiO2-Chitosan-Berbamine was shown in Figure 1(a). The CuO-TiO2-Chitosan-Berbamine nanocomposites absorbance edge was observed at 397 nm, closely related to the early literature observed CuO NPs value of 383 nm [34].Figure 1
Spectral analysis of CTCBNc. The UV-Vis spectrum (a). Spectra of photoluminescence at ambient temperature (b). FTIR investigation yielded transmittance vs. wave number chart (c).
(a)(b)(c)


### 3.2. Spectral Characteristics of CuO-TiO2-Chitosan-Berbamine Nanocomposites in the Photoluminescence (PL) Spectrum

The excitation wavelength for the PL spectrum of CuO-TiO2-Chitosan-Berbamine nanocomposites was 325 nm, as shown in Figure 1(b). At 366 nm, 424 nm, 451 nm, 481 nm, and 517 nm, the PL spectrum has five emission peaks. Recombination between electrons in the conduction band and holes in the valence band is responsible for the three near-band-edge (NBE) emissions measured at 366 nm. We observed four blue emissions at 424 nm, 451 nm, 481 nm, and 517 nm, due to the deep emission of oxygen vacancies and Cu interstitials. The 517 nm (green) wavelength of CTCBNc was generated by the recombination of a photo-generated hole with an electron in the valence band (Figure 1(b)).


### 3.3. FTIR Characteristics of CTCBNc

Figure1(c) shows the outcomes of FTIR analysis of synthesized CTCBNc. At 3434 cm-1, NH stretching bands and the wide intermolecular OH overlapped in the same area as the chitosan molecule. At 2925 cm-1 and 2865 cm-1, the asymmetric and symmetric stretching vibrations of CH3 were observed, respectively. The vibrations of both NH group vibration and the carbonyl group of the amide vibration I were found at 1639 cm-1. Chitosan molecules have antisymmetric stretching vibration peaks (-C-N and -C-O-C) at 1115 cm-1, indicating that the polysaccharide ring is associated with the molecule. For chitosan decoded with CuO/TiO2, the CH2 bending vibration was observed at 1449 cm-1 [35]. The peak C-O stretching of the berbamine characteristics was measured at 1060 cm-1 [32]. Cu-Ti-O has a metal-oxygen stretching vibration of 707, 587, and 528 cm-1 [36]. The FTIR spectrum results supported the existence of CTCBNc, the interaction of chitosan and berbamine molecules with CuO and TiO2 NPs, and the electrostatic contact between the CTCBNc surface matrix.


### 3.4. An Investigation into the Structure and Composition of CuO-TiO2-Chitosan-Berbamine Nanocomposites

Figures2(a) and 2(b), and 3 illustrate the surface morphology (FESEM/TEM) and elemental analysis (EDAX) of the synthesized CTCBNc (Figures 3a–3d). The CTCBNc produced a hexagonal-like structure, as evidenced by FESEM and TEM images. The results (Figure 3(c)) demonstrate that the edges of the hexagonal structure (chitosan and phytocompounds berbamine are coated on the hexagonal copper of metal oxide) have an average particle size of 57 nm, which is consistent with the XRD data. The selected area of the electron diffraction (SAED) pattern of prepared CuO-TiO2-Chitosan-Berbamine nanocomposites (Figure 3(d)). The EDAX spectrum of the CuO-TiO2-Chitosan-Berbamine is shown in Figure 2(b). In the CuO-TiO2-Chitosan-Berbamine nanocomposites, the atomic percentages were found to be 20.07% (C), 5.98% (N), 17.15% (Cu), 1.79% (Ti), and 55.02% (O).2Figure 2
Scanning Electron Microscope micrographs of CuO-TiO2-Chitosan-Berbamine nanocomposites at lower magnifications (a), and elemental, weight, and atomic percent compositions according to EDAX (b).
(a)(b)Figure 3
Transmission Electron Microscope images (a–c) and selected area (electron) diffraction patterns (d) of (CTCBNc).
(a)(b)(c)(d)


### 3.5. The X-Ray Diffraction Structure of Nanocomposites Composed of (CTCBNc) Nanocomposites

In Figure4(a), X-ray diffraction of synthesized CTCBNc is displayed, and the usual noncrystalline biopolymer chitosan diffraction angles, namely 2θ at 10.53° and 20.87°, are detected. The peak positions of CuO NPs at 2θ= 32.73°, 35.71°, 38.70°, 58.39°, 61.50°, and 66.24° correspond to the hkl (1 1 0), (-1 1 1), (1 1 1), (2 0 2), (-1 1 3), and (0 2 2) planes, respectively, which is compatible with the monoclinic structure of CuO NPs published in JCPDS Card (005–0661) [37]. Rutile TiO2 crystallizes in a tetragonal structure according to the XRD pattern. The diffraction peaks obtained at 2θ = 25.24°, 37.83°, 48.67°, 53.54°, 56.76°, 68.10°, and 75.22°, respectively, correspond to (101), (004), (200), (105), (211), (116), and (215) crystalline planes of rutile TiO2 (JCPDS card no. 75–1753) [38]. However, berbamine diffraction peaks were observed at 14.02°and 15.75°. The results show that the chemical synthesis of CuO-TiO2-chitosan-Berbamine is owing to steric effects and intermolecular hydrogen bonds between the CTCBNc, and the average crystallite size of the CTCBNc determined using the Debye-Scherrer formula was 57 nm [37].Figure 4
CuO-TiO2-Chitosan-Berbamine nanocomposites exhibited an X-ray diffraction pattern (a) and a distribution pattern of particle size (b).
(a)(b)


### 3.6. CTCBNc under Dynamic Light Scattering

CTCBNc has a hydrodynamic diameter of 155.20 nm, according to DLS. This is shown in Figure4(b). Due to the water surrounding the nanocomposites, the hydrodynamic diameter differs from the physical diameter.


### 3.7. Antimicrobial Activity of CTCBNc

A zone of inhibition was observed around each well containing three different concentrations (1, 1.5, and 2.0 mg/mL) of the nanocomposites against bacteria (K. pneumonia; S. pneumonia; P. vulgaris; B. subtilis) and yeast (C albicans) as shown in Figure 5. CuO-TiO2-Chitosan-Berbamine and amoxicillin produced zones with sizes ranging from 12.9 mm to 24.4 mm (Table 1). The CuO-TiO2-Chitosan-Berbamine nanocomposites show higher activity than conventional antibiotics amoxicillin because of the concentration of nanocomposites and the nature of the microorganism.Figure 5
Antimicrobial activity of CuO-TiO2-Chitosan-Berbamine nanocomposites. K pneumonia (a); S pneumonia (b); P vulgaris (c); B subtilis (d); C albicans (e).
(a)(b)(c)(d)(e)Table 1
Antimicrobial activity of CuO-TiO2-Chitosan-Berbamine nanocomposites.
Microbial nameConcentrations (mm)Positive control (mm)1 mg/mL1.5 mg/mL2 mg/mLK. pneumonia21.4 ± 0.0323.4 ± 0.0323.9 ± 0.0315.9 ± 0.03S. pneumonia17.9 ± 0.0320.5 ± 0.0321.4 ± 0.0315.0 ± 0.00P. vulgaris18.4 ± 0.0320.4 ± 0.0321.9 ± 0.0314.9 ± 0.03B. subtilis13.4 ± 0.0314.4 ± 0.0314.9 ± 0.0312.9 ± 0.03C. albicans21.9 ± 0.0323.4 ± 0.0324.4 ± 0.0315.4 ± 0.03


### 3.8. The Percentage Viability of CuO-TiO2-Chitosan-Berbamine Nanocomposites

The CuO-TiO2-Chitosan-Berbamine nanocomposites were treated with K562 cells at various concentrations for 24 hours. The CuO-TiO2-Chitosan-Berbamine nanocomposites exhibit cell death in a dose-dependent manner (Figure 6) with an IC50 of 113.54 µg/mL. The result showed a significant decrease in cell growth and percentage viability at a high dose at 24 hours.Figure 6
MTT assays resulted in cytotoxicity against T lymphoblast cells (K562) when CTCBNc was used. Three independent experiments were conducted with different concentrations of CTCBNc (6.25, 12.5, 25, 50, 100, and 200µg/mL) for 24 hours.


### 3.9. An Apoptotic Morphology Is Observed in CuO-TiO2-Chitosan-Berbamine Nanocomposites

K562 cells treated with IC50 concentrations of CTCBNc were examined by optical and fluorescence microscopy after 24 h (Figure 7). The control cells were round and crystallized, while some of the cells exposed to the drug were wrinkled and condensed (Figure 7). After staining the cells with AO and Et/Br, the control and viable cells were consistently green color (VC); early apoptosis cells had condensed and fragmented bright green chromatin (EA), and late apoptosis cells had condensed and fragmented orange chromatin (LA), under the fluorescence microscope (Figure 7). In contrast to untreated cells, the nanocomposites treated K562 cells showed more apoptotic cells, blebbing of the cell membrane, and chromatin condensation, and the observed results were similar to that of positive control drug (Doxorubicin) treated cells.Figure 7
Apoptotic cell death induced by CuO-TiO2-Chitosan-Berbamine nanocomposites in K562 blood cancer cells after 24 hours. Fluorescence microscopy (Labomed, USA) was used to analyze the cells after dual-staining with AO/EtBr (1 : 1). A) Control; B) Doxorubicin-treated cells; C) CuO-TiO2-CS-Berbamine-treated cells. LA - Late Apoptosis; EA - Early Apoptosis; NC - Necrotic Cells; VC - Viable Cells.


### 3.10. Cell Cycle Arrest Occurs in a CuO-TiO2-Chitosan-Berbamine Nanocomposites

To identify the effect of the nanocomposites, we used flow cytometry to analyze DNA content in untreated, CuO-TiO2-Chitosan-Berbamine treated K562 cells. In comparison with untreated cells, DNA content analysis of the CuO-TiO2-Chitosan-Berbamine nanocomposites-exposed K562 cells revealed a sub-G1 cell cycle. Our results showed significant cell inhibition after a treatment period of 24 hours using the selected IC50 concentration against the K562 cell line. We also used flow cytometry to determine the stages of cell cycle arrest, and the treated cells accumulated more in the sub-G1 phase of the cell cycle (Figure 8) compared to the untreated cells. Compared to untreated cells, those treated with Doxorubicin and CuO-TiO2-Chitosan-Berbamine nanocomposites at IC50 concentration showed a high percentage of cells arrested at the G2/M stage. Thus, cells cycle arrest into the G2/M phase, similar to the effect of Doxorubicin on cells (Figure 8).Figure 8
Cell cycle analysis using flow cytometry after staining with Propidium Iodide (PI). K562 cells were treated with IC50 concentration (113.54 µg/mL) of CTCBNc for 24 hours and standard drug Doxorubicin with the concentration of 5 µM/mL compared to the control. (a) Cell cycle arrest and distribution of apoptotic cells, (b) Graphical representation of the percentage of cells in each phase; ∗∗∗∗P<0.0001 untreated cells vs. CuO-TiO2-Chitosan-Berbamine nanocomposites. ∗P<0.05 untreated cells vs. CuO-TiO2-Chitosan-Berbamine nanocomposites treated cells.
(a)(b)


### 3.11. Caspase-3, 7, 8, and 9 Enzymes Are Induced in K562 Cells by Nanocomposites

The CuO-TiO2-Chitosan-Berbamine nanocomposites significantly elevated caspase-3, 7, 8, and 9 activities in comparison with untreated controls (P<0.05), whereas untreated cells did not show any activation of caspase-3, 7, 8, and 9 protein (Figure 9). Whereas positive control doxorubicin showed a higher level of caspase-3, 7, 8, and 9 protein activation, the pattern of each caspase-3, 7, 8, and 9 activation in treated cells was similar to positive control doxorubicin (Figure 9).Figure 9
Expression of Caspase 3, 7, 8, and 9 activations in apoptotic cell death using flow cytometry in K562 cells. The percentage of cells that expressed proapoptotic proteins (Caspase 3, 7, 8, and 9) in K562 cells were detected using BD FACS Calibur (BD Biosciences, CA, USA). Data are presented as the means ± SD of triplicate experiments.∗P<0.05 vs. control.


### 3.12. Nanocomposites Activate the Expression Levels of Pro- and Antiapoptotic Proteins

It has been demonstrated that the CuO-TiO2-Chitosan-Berbamine nanocomposites with IC50 concentration after 24 hours of incubation showed significant apoptosis potential in K562 cells by upregulating the expression of proapoptotic proteins Bax, Bad, Cyt C, and P53 and downregulating Bcl2 protein expression (P<0.05) (Figure 10). According to these results, CuO-TiO2-Chitosan-Berbamine nanocomposites may be therapeutically effective against human blood cancer.Figure 10
Expression of Bad, Bax, Bcl-2, p53, and Cyt C in the activation of apoptotic cell death using flow cytometry in K562 cells. Percentage of pro- and antiapoptotic proteins expressed in K562 cells. Untreated Control, Std drug (Doxorubicin 5µM/mL) treated cells, and IC50 concentration 113.54 µg/mL of CuO-TiO2-Chitosan-Berbamine nanocomposites treated cells, respectively, using BD FACS Calibur, BD Biosciences, CA, USA. Data are presented as the means ± SE of triplicate experiments. ∗P<0.05 vs. control.


### 3.13. Bax/Bcl-2 Ratio in K562 Cells

The ratio of Bax/Bcl-2 protein in CuO-TiO2-Chitosan-Berbamine nanocomposites was markedly increased as compared to untreated cells (P<0.05) (Figure 11). Treatment with doxorubicin in K562 cells significantly increased the Bax/Bcl-2 protein ratio when compared with untreated cells (P<0.05) (Figure 11).Figure 11
Bax/Bcl2 ratio in apoptotic cell death using flow cytometry in K562 cells. The values represent mean ± SD.∗p<0.05.


## 3.1. Spectral Characteristics of CuO-TiO2-Chitosan-Berbamine Nanocomposites in the UV-Visible Range

The UV-Visible absorbance spectrum of the nanocomposites CuO-TiO2-Chitosan-Berbamine was shown in Figure 1(a). The CuO-TiO2-Chitosan-Berbamine nanocomposites absorbance edge was observed at 397 nm, closely related to the early literature observed CuO NPs value of 383 nm [34].Figure 1
Spectral analysis of CTCBNc. The UV-Vis spectrum (a). Spectra of photoluminescence at ambient temperature (b). FTIR investigation yielded transmittance vs. wave number chart (c).
(a)(b)(c)


## 3.2. Spectral Characteristics of CuO-TiO2-Chitosan-Berbamine Nanocomposites in the Photoluminescence (PL) Spectrum

The excitation wavelength for the PL spectrum of CuO-TiO2-Chitosan-Berbamine nanocomposites was 325 nm, as shown in Figure 1(b). At 366 nm, 424 nm, 451 nm, 481 nm, and 517 nm, the PL spectrum has five emission peaks. Recombination between electrons in the conduction band and holes in the valence band is responsible for the three near-band-edge (NBE) emissions measured at 366 nm. We observed four blue emissions at 424 nm, 451 nm, 481 nm, and 517 nm, due to the deep emission of oxygen vacancies and Cu interstitials. The 517 nm (green) wavelength of CTCBNc was generated by the recombination of a photo-generated hole with an electron in the valence band (Figure 1(b)).


## 3.3. FTIR Characteristics of CTCBNc

Figure1(c) shows the outcomes of FTIR analysis of synthesized CTCBNc. At 3434 cm-1, NH stretching bands and the wide intermolecular OH overlapped in the same area as the chitosan molecule. At 2925 cm-1 and 2865 cm-1, the asymmetric and symmetric stretching vibrations of CH3 were observed, respectively. The vibrations of both NH group vibration and the carbonyl group of the amide vibration I were found at 1639 cm-1. Chitosan molecules have antisymmetric stretching vibration peaks (-C-N and -C-O-C) at 1115 cm-1, indicating that the polysaccharide ring is associated with the molecule. For chitosan decoded with CuO/TiO2, the CH2 bending vibration was observed at 1449 cm-1 [35]. The peak C-O stretching of the berbamine characteristics was measured at 1060 cm-1 [32]. Cu-Ti-O has a metal-oxygen stretching vibration of 707, 587, and 528 cm-1 [36]. The FTIR spectrum results supported the existence of CTCBNc, the interaction of chitosan and berbamine molecules with CuO and TiO2 NPs, and the electrostatic contact between the CTCBNc surface matrix.


## 3.4. An Investigation into the Structure and Composition of CuO-TiO2-Chitosan-Berbamine Nanocomposites

Figures2(a) and 2(b), and 3 illustrate the surface morphology (FESEM/TEM) and elemental analysis (EDAX) of the synthesized CTCBNc (Figures 3a–3d). The CTCBNc produced a hexagonal-like structure, as evidenced by FESEM and TEM images. The results (Figure 3(c)) demonstrate that the edges of the hexagonal structure (chitosan and phytocompounds berbamine are coated on the hexagonal copper of metal oxide) have an average particle size of 57 nm, which is consistent with the XRD data. The selected area of the electron diffraction (SAED) pattern of prepared CuO-TiO2-Chitosan-Berbamine nanocomposites (Figure 3(d)). The EDAX spectrum of the CuO-TiO2-Chitosan-Berbamine is shown in Figure 2(b). In the CuO-TiO2-Chitosan-Berbamine nanocomposites, the atomic percentages were found to be 20.07% (C), 5.98% (N), 17.15% (Cu), 1.79% (Ti), and 55.02% (O).2Figure 2
Scanning Electron Microscope micrographs of CuO-TiO2-Chitosan-Berbamine nanocomposites at lower magnifications (a), and elemental, weight, and atomic percent compositions according to EDAX (b).
(a)(b)Figure 3
Transmission Electron Microscope images (a–c) and selected area (electron) diffraction patterns (d) of (CTCBNc).
(a)(b)(c)(d)


## 3.5. The X-Ray Diffraction Structure of Nanocomposites Composed of (CTCBNc) Nanocomposites

In Figure4(a), X-ray diffraction of synthesized CTCBNc is displayed, and the usual noncrystalline biopolymer chitosan diffraction angles, namely 2θ at 10.53° and 20.87°, are detected. The peak positions of CuO NPs at 2θ= 32.73°, 35.71°, 38.70°, 58.39°, 61.50°, and 66.24° correspond to the hkl (1 1 0), (-1 1 1), (1 1 1), (2 0 2), (-1 1 3), and (0 2 2) planes, respectively, which is compatible with the monoclinic structure of CuO NPs published in JCPDS Card (005–0661) [37]. Rutile TiO2 crystallizes in a tetragonal structure according to the XRD pattern. The diffraction peaks obtained at 2θ = 25.24°, 37.83°, 48.67°, 53.54°, 56.76°, 68.10°, and 75.22°, respectively, correspond to (101), (004), (200), (105), (211), (116), and (215) crystalline planes of rutile TiO2 (JCPDS card no. 75–1753) [38]. However, berbamine diffraction peaks were observed at 14.02°and 15.75°. The results show that the chemical synthesis of CuO-TiO2-chitosan-Berbamine is owing to steric effects and intermolecular hydrogen bonds between the CTCBNc, and the average crystallite size of the CTCBNc determined using the Debye-Scherrer formula was 57 nm [37].Figure 4
CuO-TiO2-Chitosan-Berbamine nanocomposites exhibited an X-ray diffraction pattern (a) and a distribution pattern of particle size (b).
(a)(b)


## 3.6. CTCBNc under Dynamic Light Scattering

CTCBNc has a hydrodynamic diameter of 155.20 nm, according to DLS. This is shown in Figure4(b). Due to the water surrounding the nanocomposites, the hydrodynamic diameter differs from the physical diameter.


## 3.7. Antimicrobial Activity of CTCBNc

A zone of inhibition was observed around each well containing three different concentrations (1, 1.5, and 2.0 mg/mL) of the nanocomposites against bacteria (K. pneumonia; S. pneumonia; P. vulgaris; B. subtilis) and yeast (C albicans) as shown in Figure 5. CuO-TiO2-Chitosan-Berbamine and amoxicillin produced zones with sizes ranging from 12.9 mm to 24.4 mm (Table 1). The CuO-TiO2-Chitosan-Berbamine nanocomposites show higher activity than conventional antibiotics amoxicillin because of the concentration of nanocomposites and the nature of the microorganism.Figure 5
Antimicrobial activity of CuO-TiO2-Chitosan-Berbamine nanocomposites. K pneumonia (a); S pneumonia (b); P vulgaris (c); B subtilis (d); C albicans (e).
(a)(b)(c)(d)(e)Table 1
Antimicrobial activity of CuO-TiO2-Chitosan-Berbamine nanocomposites.
Microbial nameConcentrations (mm)Positive control (mm)1 mg/mL1.5 mg/mL2 mg/mLK. pneumonia21.4 ± 0.0323.4 ± 0.0323.9 ± 0.0315.9 ± 0.03S. pneumonia17.9 ± 0.0320.5 ± 0.0321.4 ± 0.0315.0 ± 0.00P. vulgaris18.4 ± 0.0320.4 ± 0.0321.9 ± 0.0314.9 ± 0.03B. subtilis13.4 ± 0.0314.4 ± 0.0314.9 ± 0.0312.9 ± 0.03C. albicans21.9 ± 0.0323.4 ± 0.0324.4 ± 0.0315.4 ± 0.03


## 3.8. The Percentage Viability of CuO-TiO2-Chitosan-Berbamine Nanocomposites

The CuO-TiO2-Chitosan-Berbamine nanocomposites were treated with K562 cells at various concentrations for 24 hours. The CuO-TiO2-Chitosan-Berbamine nanocomposites exhibit cell death in a dose-dependent manner (Figure 6) with an IC50 of 113.54 µg/mL. The result showed a significant decrease in cell growth and percentage viability at a high dose at 24 hours.Figure 6
MTT assays resulted in cytotoxicity against T lymphoblast cells (K562) when CTCBNc was used. Three independent experiments were conducted with different concentrations of CTCBNc (6.25, 12.5, 25, 50, 100, and 200µg/mL) for 24 hours.


## 3.9. An Apoptotic Morphology Is Observed in CuO-TiO2-Chitosan-Berbamine Nanocomposites

K562 cells treated with IC50 concentrations of CTCBNc were examined by optical and fluorescence microscopy after 24 h (Figure 7). The control cells were round and crystallized, while some of the cells exposed to the drug were wrinkled and condensed (Figure 7). After staining the cells with AO and Et/Br, the control and viable cells were consistently green color (VC); early apoptosis cells had condensed and fragmented bright green chromatin (EA), and late apoptosis cells had condensed and fragmented orange chromatin (LA), under the fluorescence microscope (Figure 7). In contrast to untreated cells, the nanocomposites treated K562 cells showed more apoptotic cells, blebbing of the cell membrane, and chromatin condensation, and the observed results were similar to that of positive control drug (Doxorubicin) treated cells.Figure 7
Apoptotic cell death induced by CuO-TiO2-Chitosan-Berbamine nanocomposites in K562 blood cancer cells after 24 hours. Fluorescence microscopy (Labomed, USA) was used to analyze the cells after dual-staining with AO/EtBr (1 : 1). A) Control; B) Doxorubicin-treated cells; C) CuO-TiO2-CS-Berbamine-treated cells. LA - Late Apoptosis; EA - Early Apoptosis; NC - Necrotic Cells; VC - Viable Cells.


## 3.10. Cell Cycle Arrest Occurs in a CuO-TiO2-Chitosan-Berbamine Nanocomposites

To identify the effect of the nanocomposites, we used flow cytometry to analyze DNA content in untreated, CuO-TiO2-Chitosan-Berbamine treated K562 cells. In comparison with untreated cells, DNA content analysis of the CuO-TiO2-Chitosan-Berbamine nanocomposites-exposed K562 cells revealed a sub-G1 cell cycle. Our results showed significant cell inhibition after a treatment period of 24 hours using the selected IC50 concentration against the K562 cell line. We also used flow cytometry to determine the stages of cell cycle arrest, and the treated cells accumulated more in the sub-G1 phase of the cell cycle (Figure 8) compared to the untreated cells. Compared to untreated cells, those treated with Doxorubicin and CuO-TiO2-Chitosan-Berbamine nanocomposites at IC50 concentration showed a high percentage of cells arrested at the G2/M stage. Thus, cells cycle arrest into the G2/M phase, similar to the effect of Doxorubicin on cells (Figure 8).Figure 8
Cell cycle analysis using flow cytometry after staining with Propidium Iodide (PI). K562 cells were treated with IC50 concentration (113.54 µg/mL) of CTCBNc for 24 hours and standard drug Doxorubicin with the concentration of 5 µM/mL compared to the control. (a) Cell cycle arrest and distribution of apoptotic cells, (b) Graphical representation of the percentage of cells in each phase; ∗∗∗∗P<0.0001 untreated cells vs. CuO-TiO2-Chitosan-Berbamine nanocomposites. ∗P<0.05 untreated cells vs. CuO-TiO2-Chitosan-Berbamine nanocomposites treated cells.
(a)(b)


## 3.11. Caspase-3, 7, 8, and 9 Enzymes Are Induced in K562 Cells by Nanocomposites

The CuO-TiO2-Chitosan-Berbamine nanocomposites significantly elevated caspase-3, 7, 8, and 9 activities in comparison with untreated controls (P<0.05), whereas untreated cells did not show any activation of caspase-3, 7, 8, and 9 protein (Figure 9). Whereas positive control doxorubicin showed a higher level of caspase-3, 7, 8, and 9 protein activation, the pattern of each caspase-3, 7, 8, and 9 activation in treated cells was similar to positive control doxorubicin (Figure 9).Figure 9
Expression of Caspase 3, 7, 8, and 9 activations in apoptotic cell death using flow cytometry in K562 cells. The percentage of cells that expressed proapoptotic proteins (Caspase 3, 7, 8, and 9) in K562 cells were detected using BD FACS Calibur (BD Biosciences, CA, USA). Data are presented as the means ± SD of triplicate experiments.∗P<0.05 vs. control.


## 3.12. Nanocomposites Activate the Expression Levels of Pro- and Antiapoptotic Proteins

It has been demonstrated that the CuO-TiO2-Chitosan-Berbamine nanocomposites with IC50 concentration after 24 hours of incubation showed significant apoptosis potential in K562 cells by upregulating the expression of proapoptotic proteins Bax, Bad, Cyt C, and P53 and downregulating Bcl2 protein expression (P<0.05) (Figure 10). According to these results, CuO-TiO2-Chitosan-Berbamine nanocomposites may be therapeutically effective against human blood cancer.Figure 10
Expression of Bad, Bax, Bcl-2, p53, and Cyt C in the activation of apoptotic cell death using flow cytometry in K562 cells. Percentage of pro- and antiapoptotic proteins expressed in K562 cells. Untreated Control, Std drug (Doxorubicin 5µM/mL) treated cells, and IC50 concentration 113.54 µg/mL of CuO-TiO2-Chitosan-Berbamine nanocomposites treated cells, respectively, using BD FACS Calibur, BD Biosciences, CA, USA. Data are presented as the means ± SE of triplicate experiments. ∗P<0.05 vs. control.


## 3.13. Bax/Bcl-2 Ratio in K562 Cells

The ratio of Bax/Bcl-2 protein in CuO-TiO2-Chitosan-Berbamine nanocomposites was markedly increased as compared to untreated cells (P<0.05) (Figure 11). Treatment with doxorubicin in K562 cells significantly increased the Bax/Bcl-2 protein ratio when compared with untreated cells (P<0.05) (Figure 11).Figure 11
Bax/Bcl2 ratio in apoptotic cell death using flow cytometry in K562 cells. The values represent mean ± SD.∗p<0.05.


## 4. Discussion

Chronic Myelogenous Leukaemia (CML) is a rare type of bone marrow cancer. The quantity of white blood cells in the blood increases as a result of CML. Several advances in treatment have improved the prognosis for people with CML [39]. A human cell has 23 pairs of chromosomes by default, and in chronic myelogenous leukemia, the chromosomes are arranged in a different order. This creates an extrashort chromosome called the Philadelphia chromosome, and 90% of people with this type of leukemia have this chromosome in their blood cells [40]. Through the use of nanotechnology in drug delivery for cancer therapy, easy attachment to cancerous cells can be achieved selectively. It is possible to program nanoparticles, such as gold, to identify cancerous cells and deliver drugs selectively and precisely, preventing interference with normal cells.Because of their biological properties, nanocomposites are known for their inherent anticancer properties. The intrinsic features of molecularly generated nanocomposite materials, such as disruptions in normal cell cycle operations, interference with DNA, RNA, protein synthesis, and hormone disruption, are demonstrated to impede the growth of cancer cells [41]. As a systemic therapy, nanocomposites have the ability to interact with veins and arteries as well as the stromal tissues surrounding tumors, preventing their growth with minimal side effects [42].As shown in Figure1(a), FTIR was used to investigate and determine CuO-TiO2-Chitosan-Berbamine nanocomposites, with an absorption band ranging from 250-480 nm (the wavelength of CuO-Chitosan nanocomposites) [43]. In our study, we identified that nanoparticles were grouped by properties of similar size and shape, and they were distributed consistently, according to SEM analysis (Figures 2(a) and 2(b)). The purity and stoichiometry of nanocomposites during the synthesis process were made more accessible with EDS analysis. No impurity peaks are visible in the EDS pattern, indicating the occurrence of O, N, Cu, and C elements (Figures 2(a) and 2(b)). The dispersion of each element was calculated using the appropriate K-line from the X-ray spectra (O, N, Cu, and C). As the nanocomposites percentage increases, there are some localized accumulations of nanocomposites visible in the EDS plotting images. The residual peaks are indexed to the hexagonal structure of the nanocomposites, which matches the standard data (Figures 2(a) and 2(b)). XRD data showed that the precursor molecules had transformed into nanocomposites after calcination. The strong and sharp peaks indicate well-ordered crystalline samples (Figure 4(a)).In K562 cancer cells, (CTCBNc) nanocomposites significantly improve apoptotic cell death via causing cytotoxicity, as revealed in this study (Figures5 and 6). A previous study found that berbamine nanoparticles downregulated the BCR/ABL gene and induced an antileukemic effect in K562 cells [44]. In numerous studies, copper-based nanoparticles have been shown to cause apoptosis only in cancerous cells, not in normal ones [45]. Nano-sized compounds have been linked to increased intracellular ROS and cytotoxicity in several studies, and as a growth and development stimulator in both conditions, the cytosolic reactive oxygen species can play various roles. Under stressful situations, however, it may also cause cell death [46, 47]. CuO-TiO2-Chitosan-Berbamine nanocomposites trigger the apoptotic process by generating high levels of intracellular ROS (Figure 7) and paying attention to the depolarized mitochondrial membrane is vital due to the weakening of responsive genes (Figure 7).The potential applications of hybrid nanocomposites have sparked a lot of interest in the last decade, particularly CS-TiO2 composites. These composites have fascinating properties due to the combination and additive properties of organic and inorganic materials [48]. Previous research has demonstrated the antimicrobial properties and antiproliferative activity of CS-TiO2 NPs [49, 50] and also discovered a CS-TiO2 NPs-based food preservation film and methyl orange dye degrading CS-TiO2 composites [51]. The CS-TiO2 nanoparticles may be used for industrial wastewater treatment because the functionalized CS possesses adsorbent properties such as mesoporous properties, which enhance CS-TiO2 interactions, nano-size, and surface area [52].In zones around the well loaded with different concentrations (1, 1.5, and 2 mg/mL) of nanocomposites, the fungalC. albicans, as well as both gram-positive (B. subtilis and S. pneumoniae) and gram-negative (P. vulgaris and K. pneumoniae) bacterial strains were inhibited. CuO-TiO2-Chitosan-Berbamine and amoxicillin zone sizes ranged from 12.9 mm to 24.4 mm (Table 1). Based on the concentration of nanocomposites and the type of target cells, the CuO-TiO2-Chitosan-Berbamine has been found to be more effective than the conventional antibiotic amoxicillin. Several factors influence the activity of berbamine as an antimicrobial agent. As a result of oxidative stress caused by CuO-TiO2-Chitosan-Berbamine, free radicals (ROS) are generated in microbial or cancer cells. The release of Ti2+/Cu2+ ions, the aptitude of the chitosan to diffuse, the surface-volume ratio, particle size, electrostatic attraction, and the increased surface defects which leads to vacancies of oxygen can all cause oxidative stress inside cells, which can lead to antimicrobial activity [53]. Green emission in the PL spectrum occurs as a result of a single ionized oxygen vacancy. The presence of oxygen vacancies on the surface of CuO-TiO2-Chitosan-Berbamine is a critical factor affecting biocidal activity. Production of singlet oxygen and hydroxide radicals inside cells due to both an increase in surface defects, including oxygen vacancies, and a split-water mechanism that allows ROS and other active free radicals [54].In this study, the viability of the cells was resolved by the MTT assay. The CuO-TiO2-Chitosan-Berbamine nanocomposites caused cell death in a dose-dependent manner, with an IC50 of 113.54 µg/mL (Figure 6). The high-dose result revealed a significant increase in cell growth and viability after 24 hours. In some places, the drug-exposed cells were wrinkled and condensed, whereas the control cells were round and crystallized (Figure 7). The control and viable cells had a normal green color after staining with AO and Et/Br, while early apoptosis cells had condensed and fragmented bright green chromatin, and late apoptosis cells had condensed and fragmented orange chromatin under the fluorescence microscope (Figure 7). Similar results were reported in previous studies of K562 cells, including apoptosis and membrane blebbing, as well as chromatin condensation, in comparison to untreated cells [55, 56].The percentage of cells that were arrested at several stages of the K562 cell cycle is shown in the results. Untreated, Standard, and CuO-TiO2-Chitosan-Berbamine nanocomposites with IC50 concentration arrested 2.38%, 3.45%, and 2.45% of cells in the Sub G0/G1 phase (Apoptotic phase). Treatment-free, Standard, and CuO-TiO2-Chitosan-Berbamine nanocomposites with IC50 concentration arrested 70.25%, 30.28%, and 56.82% of cells in the G0/G1 phase (Growth Phase). Untreated, Standard, and CuO-TiO2-Chitosan-Berbamine nanocomposites with IC50 concentration arrested 8.20%, 24.65%, and 10.22% of cells in the S phase (synthetic phase). In the G2/M phase, however, 18.38%, 39.39%, and 28.36% of cells in Untreated, Standard, and CuO-TiO2-Chitosan-Berbamine nanocomposites with IC50 concentrations were arrested, respectively (Figure 8). When compared to control cells, cells treated with doxorubicin and CuO-TiO2-Chitosan-Berbamine nanocomposites at IC50 concentration have a high percentage of cells in the G2/M stage. As a result, the cell cycle was arrested at the G2/M stage. In K562 cells, the nanocomposites caused a significant cell cycle phase arrest, similar to doxorubicin. An earlier study found that Jellyfish-HE's anticancer activity resulted from caspase, MAPK activation, and cell cycle arrest at the G1/S phase of the cell cycle in K562 cells [57].The results suggest that the CuO-TiO2-Chitosan-Berbamine nanocomposites with the IC50 concentration after a 24-hour incubation period showed significant apoptotic potential on K562 cells, with significantly upregulated expression of death-associated proteins, Caspase 3, -7, -9, -8, Bad, Cyt C, Bax, and P53 and significantly downregulated expression of Bcl2. Caspases, which inhibit the production of cancer-responsive genes, are activated when the mitochondrial membrane is disrupted. In comparison to control K562 cells, treatment with IC50 concentrations of CTCBNc activated apoptosis which leads to cell death via a mitochondrial-dependent pathway (Figures 9 and 10).It has also been demonstrated that caspases, which are aspartic acid-degrading enzymes, cause apoptosis by decreasing cellular resistance to apoptotic stimuli [58, 59]. In addition to caspases, other mechanisms play a part in apoptosis [60]. We report that CuO-TiO2-Chitosan-Berbamine nanocomposites activate caspases 3, 7, 8, and 9 in K562 cells, triggering apoptosis both in intrinsic and extrinsic pathways. Nanocrystals of CuO-C showed much less toxicity toward cancer cells than synthesized Cu nanoparticles [61]. Human K562 cells were treated with nanocomposites composed of CuO-TiO2-Chitosan-Berbamine that had enhanced apoptotic potency and specificity. Inhibitory properties were observed in polymer conjugated (CTCBNc) nanocomposites due to their ability to promote apoptosis, decrease metastatic growth, and possibly circumvent antibiotic resistance.


## 5. Conclusion

The CuO-TiO2-Chitosan-Berbamine nanocomposites demonstrate the potential of traditional ethnic medicines to yield new nanomaterial-based drugs. Conforming to our outcomes, the CuO-TiO2-Chitosan-Berbamine nanocomposites induce mitochondrial apoptosis by targeting proteins from the BCL-2 family (i.e., BAX and BCL-2). In K562 cells, CuO-TiO2-Chitosan-Berbamine nanocomposites-induced apoptosis was accompanied by an increase in p53 and Bax levels and caspase activation. CuO-TiO2-Chitosan-Berbamine nanocomposites may enhance cellular defense mechanisms against Chronic Myelogenous Leukemia if it is included in pharmaceutical or cosmeceutical formulations. However, more investigations need to be done to develop a feasible drug formulation with CuO-TiO2-Chitosan-Berbamine nanocomposites.



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