Synthesis and characterization of ZnO – TiO 2 – chitosan – escin metallic nanocomposites: Evaluation of their antimicrobial and anticancer activities

: This work intended to formulate bio - nanocom posites of zinc oxide ( ZnO ) , titanium oxide ( TiO 2 ) , chitosan, and escin, characterize their physical properties, and eval uate their antimicrobial and anticancer properties. X - ray di ﬀ ractometers ( XRD ) and scanning and transmission electron microscopes were applied to characterize the mor phology and ultrastructure of chemically synthesized bio - nanocomposites. To investigate the functional groups of bio - nanocomposites, we used Perkin – Elmer spectro meters for Fourier transform infrared ( FTIR ) analysis and photoluminescence ( PL ) spectroscopy for PL spectrum analysis. Antimicrobial activities against bacterial and fungal strains were tested with agar well di ﬀ usion. Bio nanocomposites were tested for anticancer e ﬀ ects on a MOLT4 blood cancer cell line using morphological ana lysis, methyl thiazole tetrazolium assay, apoptosis by acridine orange/ethidium bromide, and mitochondrial mem brane potential ( ΔΨ m ) . In XRD, FTIR, and PL, the active compounds of ZnO – TiO 2 , chitosan, and escin peaks were observed. Our bio - nanocomposites demonstrated antimicro bial activity against bacterial and fungal pathogens. The bio nanocomposite was cytotoxic to MOLT4 cells at an IC 50 concentration of 33.4 µg · mL − 1 . Bio - nanocomposites caused cytotoxicity, changes in cell morphology, and mitochondrial membrane potential degradation, all of which resulted in apop totic cell death. MOLT4 cells were found to be responsive to bio nanocomposites based on ZnO – TiO 2 – chitosan – escin.


Introduction
The high mortality rate is still recorded due to emerging and re-emerging diseases despite major leaps in the development of pharmaceutical drugs to treat a range of infectious or non-infectious diseases [1]. The development of effective and inexpensive drugs is aimed at limiting the economic and social burden of these diseases. Nanotechnology has gained attention in a variety of fields, including bioengineering, medical applications, clinical drug development, and imaging studies [2,3]. Notably, the nanotechnology platform has positively contributed to the treatment of rare disease conditions such as hereditary transthyretin amyloidosis via siRNA-based delivery to silent the gene responsible for the disease [4]. Similarly, in recent days, numerous researchers have focused on developing nanoparticle or nanocomposite-based drugs and testing their efficacy in various human disease conditions including cancer [5], diabetes [6], and neurodegenerative conditions [7]. Indeed, the investigation of nanoforms of plant-derived phytochemicals has gained significant attention to develop drugs for cancer and various other human diseases affecting the communities [8].
Leukemia comprises malignancies of blood cells derived from blood-forming tissues in the bone marrow [9]. In the past three decades, the incidence of leukemia has risen rapidly, and the demand for drug resources to combat it is highly warranted [10]. Although many drugs have been developed and tested in leukemic patients, the treatment remains challenging and mortality is inevitable [11], mainly due to the development of resistance to drug treatments and metastasis of leukemic cells [12]. Notably, the progression of drug resistance in leukemic cells was found to originate from leukemia stem cells (LSCs). Several factors responsible for the failure to target drug-resistant leukemia cells with drug formulations have been reviewed in detail [13,14]. Currently, to test the efficacy of new drug candidates against leukemia, a human T lymphoblast cell line also known as high migrating drug-surviving MOLT4 cells has been used across various laboratories in leukemia research [15,16].
Escin is a phytochemical constituent of the traditional medicinal plant Aesculus hippocastanum (horse chestnut) [17]. It has been used to treat rectal hemorrhoids and hematomas and exhibits other pharmacological benefits like anti-inflammatory, anticancer, and cardioprotective properties in diabetic experimental models [18][19][20]. This study has characterized the physicochemical of a newly synthesized drug candidate using Zn, TiO 2 , and chitosan conjugated with escin and identified its potential as an antimicrobial and in vitro anti-tumoral agent on the MOLT4 blood cancer. For the latter purpose, the ability of synthesized nanoparticles to induce apoptosis and increase reactive oxygen species (ROS) production in MOLT4 cells will be assessed.

Bacterial growth inhibitory studies
A well diffusion approach was utilized to investigate the antibacterial capacity of the formulated ZnO-TiO 2 -chitosan-escin bio-nanocomposite. Bacterial strains from Gram-positive strains (S. aureus and B. subtilis) and Gram-negative strains (P. aeruginosa and E. coli) were employed. In this investigation, strains were inoculated individually on sterile Mueller Hinton agar containing Petri discs. The antibacterial activity of a ZnO-TiO 2 -chitosan-escin bio-nanocomposites at 1, 1.5, and 2 mg·mL −1 dispersed in a 5% sterile dimethyl sulfoxide (DMSO) solution was demonstrated. The plates were maintained for 24 h at 37°C, and then inhibition zones were determined after 24 h. Amoxicillin antibiotic (30 µg·mL −1 ) was utilized as positive drug, and the tests were performed in triplicates.

Antifungal properties
The antifungal potential of Candida albicans was assessed using standard procedures as reported earlier and growth on PDA [23]. The C. albicans strain was injected onto the PDA agar petri plate by streaking twice or thrice, followed by moving the plates in a circular plane at an angle of 60°for each streak to ensure homogeneity in the inoculums spreading. Following that, germ-free forceps were used to put wells containing 1, 1.5, and 2 µg·mL −1 of test ZnO-TiO 2 -chitosan-escin bio-nanocomposite onto the inoculation plates, which were then sustained at 30°C for 24 h under visible light. Following the incubation of C. albicans with synthesized bio-nanocomposites, the inhibition zones were determined to assess the growth inhibitory efficacy of ZnO-TiO 2 -chitosan-escin bionanocomposite. The amoxicillin (30 µg·mL −1 ) was employed as a positive control. Experiments were performed in triplicates.

Cell culture and maintenance
MOLT4 cells were grown in a humidified room using RPMI-1640 medium enriched with 10% of FBS and 1% of penicillin-streptomycin antibiotic. Subcultured cells were loaded at the population of around 4 × 10 5 cells·mL −1 . As soon as the cells reached confluence, they were immediately subcultured into fresh cell culture dishes according to the experiment's requirements. MOLT4 cells were fed fresh RPMI-1640 media every 2-3 days, depending on the cell density.

Methyl thiazole tetrazolium assay for cell viability
Cytotoxicity of the synthesized bio-nanocomposites on the MOLT4 cancer cells was carried out by using MTT assay. MOLT4 cells were first grown on 96-well clearbottom plates at a population of 1 × 10 4 cells·well −1 . After 24 h, the MOLT4 cells were administered with bionanocomposites at various doses (10, 20, 30, 40, 50, and 60 µg·mL −1 ) for 72 h. Following the treatment period, cells were cleansed with sterile PBS, and 20 µL of MTT was mixed in each well and further sustained for 4 h, with regular monitoring under an inverted microscope for the production of blue-colored formazan crystals. After the development of formazan stones, the media was removed from wells and added with 150 µL of DMSO to solubilize the formazan. The plates were rotated on an orbital shaker to obtain a uniform solution. The OD was quantified using a plate reader at 570 nm. MTT assay was performed following procedures reported previously and with required modifications for our study [24]. The results of OD were utilized to determine the IC 50 values, and the percentage of cell death is quantified using the formula: (1)

Cell morphological analysis and staining with AO/EtBr
analysis, the cells were immobilized using 4% PFA in PBS for 10 min. The morphological alterations were detected using an inverted phase-contrast microscope (Olympus Corporation). The PBS-washed MOLT4 blood cancer cells were also incubated using 1 µM AO/EtBr at 37°C for 15 min under dark conditions. Finally, the stained MOLT4 cells were observed and documented using a fluorescent microscope for the visualization of cells stained with the AO/EtBr dyes. Experiments were performed in triplicates.

Measurement of MMP level (ΔΨm)
MOLT4 cells were cultured onto the 24-well plate at a population of 2 × 10 5 cells·well −1 and treated for 24 h with IC 50 concentrations (30 and 40 µg·mL −1 ) of the bionanocomposite. The state of MMP was evaluated using the rhodamine-123 (Rh-123). Rh-123 penetrates the mitochondrial matrix and requires mitochondrial transmembrane potential to induce PL. MOLT4 cells were treated with 10 mM Rh 123 for 30 min before being cleansed thrice with PBS. Finally, the intensity of fluorescence was detected using a spectrofluorometer at 488 and 510 nm, respectively, as excitation and emission wavelengths. The fraction of control cells with MMP was calculated.

Nuclear fluorescence labeling on MOLT4 cells to detect nuclear damage
The morphology of nucleic acid material was examined using fluorescence microscopy using the morphology of nucleic aci staining technique [25]. A population of 1 × 10 4 cells·well −1 was used for the seeding of MOLT4 cells in the bottom dish. An atmosphere of 5% CO 2 and 37°C humidified for 24 h was used for incubation. Incubated for another 24 h under the same conditions, the cells were then exposed to ZnO-TiO 2 -chitosan-escin bio-nanocomposite concentrations (30 g and 40 µg·mL −1 ) except for the control group. A DAPI solution (diluted in methanol) was added after the cells had been washed twice with 1× PBS. The plates were enclosed in aluminum foil to prevent light exposure and incubated for a further 15 min. Fluorescence microscopy was used to examine the nuclear material morphology (Biorad, ZOE fluorescent imaging system, Germany).

An assessment of ROS accumulation
To estimate the ROS accumulation using fluorescence microscopy, the dichloro-dihydro-fluorescin diacetate (DCFH-DA) staining technique was used. The MOLT4 cells were treated for 24 h with 30 and 40 µg of ZnO-TiO 2 -chitosan-escin bio-nanocomposite, and the untreated cells served as controls. Roughly 2 × 10 5 of cells in 1 mL suspension was mixed with 10 µM DCFH-DA solution and allowed to incubate for 5 min at 37°C. After washing and resuspending the cells in PBS solution, the fluorescence images were acquired using a fluorescent microscope at 485 and 530 nm, respectively, as excitation and emission wavelengths.

Statistical analysis
Data were illustrated as mean ± SD, and the variance between the groups was calculated using a one-way ANOVA and DMRT tests. Statistically significant was determined as the p-value was less than 0.05.

Characterization of ZnO-TiO 2 -chitosan-escin bio-nanocomposites
ZnO-TiO 2 -chitosan-escin bio-nanocomposites show X-ray diffraction characteristics in Figure 1. According to the XRD molecular patterns, ZnO was the major face found at angles  (Figure 1), the average crystallite size of bio-nanocomposites is 55 nm.
A FESEM analysis and TEM analysis of a ZnO-TiO 2chitosan-escin bio-nanocomposite ( Figure 2) and TEM analysis ( Figure 3) were carried out to determine its surface morphology. ZnO, TiO 2 , chitosan, and escin bio-nanocomposites were visualized using FESEM and TEM images, showing a nanorod structure with an average particle  In the ZnO-TiO 2 -chitosan-escin samples, the FTIR spectrum revealed the presence of ZnO, TiO 2 , chitosan, and escin functional groups. The broad -OH and -NH peaks with hydrogen bonds were detected in the chitosan peaks at 3,419 and 1,608 cm −1 , showing the amide I group (C-O stretching combined with the N-H deformation mode) (Figure 4). During the carbonic acid salt's stretching vibration, the COO − group produced the peak at 1,384 cm −1 , and during the glucose circle's stretching vibration, the peak at 1,066 cm −1 was caused by the stretching vibration of C-O-C [26]. Escin's distinctive summits, on the other hand, are as follows: There were asymmetric and symmetric peaks in C-H group at 2,924 and 2,846 cm −1 , as well as C-O stretching and C-H out-of-plane bending peaks at 1,161 and 934 cm −1 [27]. The metal-oxygen (M-O) stretching vibration, similar to Zn-Ti-O, was discovered at 718 and 532 cm −1 [28]. The results of the FTIR spectrum suggest that the bio-nanocomposite performed effectively with the chitosan and escin molecules in the ZnO and TiO 2 surface matrices. These interactions were caused by the electrostatic interaction between the ZnO-TiO 2 -chitosan-escin bio-nanocomposite (Figure 4).
Ultraviolet-Visible absorbance spectrum of the ZnO-TiO 2 -chitosan-escin bio-nanocomposite dispersed in water is shown in Figure 4. The UV absorbance edge peak was centered at 370 nm for hexagonal wurtzite ZnO-TiO 2 -chitosan-escin bio-nanocomposite [29]. The redshift (5 nm) was observed at the synthesized ZnO-TiO 2 -chitosan-escin bio-nanocomposite absorbance peak as compared to that of bulk ZnO (365 nm) [30]. Because of foreign (TiO 2 -chitosan-escin) molecules on the lattice surface, this redshift can be attributed to the development of shallow levels inside the bandgap [31]. The PL spectra of a synthesized ZnO-TiO 2 -chitosan-escin bio-nanocomposite with an excitation wavelength of 325 nm are revealed in Figure 4. The ZnO-TiO 2 -chitosan-escin bio-nanocomposite PL emission spectrum was found at 379, 410, 420, 441, 458, 476, and 500 nm, respectively ( Figure 4). Near band edge emission at 379 nm was found, corresponding to the radiative recombination of the free exciton-exciton collision mechanism [32]. The violet emission seen at 410 nm could be caused by electron transfer from a surface donor level of zinc interstitials (Zn i ) to the top level of the valence band [26]. The three blue emission bands at 420, 441, 458, and 476 nm [26] represent singly ionized Zn vacancies (VZn). The green emission band at 500 nm could be caused by oxygen vacancies (O v ) [26].

ZnO-TiO 2 -chitosan-escin bio-nanocomposite-induced antimicrobial activity
The antibacterial efficacy of the produced ZnO-TiO 2 -chitosanescin bio-nanocomposite was investigated using an agar well diffusion approach against (S. aureus and B. subtilis) as a gram-positive and (P. aeruginosa and E. coli) as a gram-negative bacterial strains and fungal Candida albicans infections ( Figure 5). ZnO-TiO 2 -chitosan-escin caused a zone of inhibition against gram-positive, gram-negative, and fungal strains, as shown in Figure 5.
According to the findings, raising the concentration of ZnO-TiO 2 -chitosan-escin bio-nanocomposite increased the widths of the inhibitory zones for all of the tested pathogens.

ZnO-TiO 2 -chitosan-escin bio-nanocomposite-induced cytotoxicity on MOLT4 cells
We performed an MTT assay to investigate the impacts of bio-nanocomposites on the MOLT4 cell line. In this study, the bio-nanocomposites exhibited cytotoxicity on MOLT4 cells at a 33.4 µg·mL −1 (IC 50 ) dose. Therefore, for all subsequent experiments testing its anticancer effect, we treated cells for 24 h with 30 and 40 µg·mL −1 , following which we collected the drug-surviving cells ( Figure 6).

Bio-nanocomposite-induced morphological changes in MOLT4 cells
Bio-nanocomposites were added to MOLT4 cells at a dose of 30 and 40 µg·mL −1 and incubated for 24 h. Following

ZnO-TiO 2 -chitosan-escin bio-nanocomposites-induced apoptosis in MOLT4 cells
Following a 24-h treatment with the bio-nanocomposites at a dose of 30 and 40 µg·mL −1 , MOLT4 cells were fixated, stained with AO/EtBr, and monitored using a fluorescence microscope. A green fluorescence signal indicating alive cells and free of apoptosis was detected by AO in the control cells; however, yellowish orange fluorescence was detected in the nanocomposite-treated cells, indicating earlier stage and late stage of apoptotic cell death, and condensation of nucleic acid and red fluorescence indicate the cell death was carried out by necrosis, respectively ( Figure 8). The fluorescence intensity increased as the dosage of the nanocomposite increased ( Figure 8). As a result, the capacity of nanocomposite to cause apoptosis in blood cancer cells became apparent.

ZnO-TiO 2 -chitosan-escin bio-nanocomposites-induced nuclear changes in MOLT4 cells
By interacting with the cells, ZnO-TiO 2 -chitosan-escin bio-nanocomposites altered cell permeability, aggregated, disrupted enzyme systems, and produced ROS. DAPI staining revealed intact nuclear morphology in the control panel, but condensed or fragmented morphology in treatment-treated cells (Figure 9). Morphological indicators of apoptosis, such as fragmented and condensed DNA, indicated the onset of cell death. The inhibition of cell cycle progression, the disruption of cell permeability, the accumulation of ROS, and stimulation of the caspase cascade all slowed down cell growth.  MOLT4 cells treated with the nanocomposites showed dull or decreased green fluorescence, indicating that the cells' MMP was inhibited. This discovery indicated that nanocomposite could reduce MMP levels in MOLT4 cells, explaining the higher ROS levels ( Figure 10).

ZnO-TiO 2 -chitosan-escin bio-nanocomposites-induced production of ROS in MOLT4 cells
We used DCFH-DA staining to assess ROS expression in untreated cells and compared the results with the bionanocomposites-treated MOLT4 cells. The intensity of the bright fluorescent green color can be considered proportional to the level of ROS produced by the cells (Figure 11). In the experimental cell group treated with bio-nanocomposites at 40 µg·mL −1 , there was a greater expression of ROS. In the group of cells treated with bio-nanocomposites at 30 µg·mL −1 , ROS expression was remarkably reduced, whereas ROS was almost negligible in control cells.

Discussion
The requirement for effective drug candidates for various diseases of humans and plant species is in growing demand [33]. In recent days, due to the development of drug resistance, many effective drugs for infectious diseases [34] and cancer have become ineffective and turned out to be an economic loss for the pharmaceutical industries and worrisome for research communities [35]. In our study, we have synthesized the bio-nanocomposite drugusing bimetallic complex zinc (Zn) and titanium (TiO 2 ) complexed with chitosan and conjugated with a plant phytochemical escin, a component of traditional plant  medicine Aesculus hippocastanum. Following the production of bio-nanocomposites, we have characterized the synthesized bio-nanocomposites to confirm their physical characteristics. Analysis using XRD, DLS, FESEM, TEM, EDAX spectrum, FTIR, and UV absorbance analysis all showed typical characteristics of a bio-nanocomposite as reported earlier with similar metallic complex and a plant phytoconstituents [36].
We additionally tested the bio-nanocomposites for antimicrobial potential against P. aeruginosa, E. coli, S. aureus, and B. subtilis bacteria and Candida albicans pathogen, using the agar well diffusion method. Our results showed escin bio-nanocomposites showed significant antibacterial and antifungal activity. The zone of inhibitions in antibacterial and antifungal analyses was significantly comparable to standard antibiotic treatments using amoxicillin. In our study, the observed antibacterial activity of bio-nanocomposites could be accredited to various causes, comprising: (1) the accumulation of bionanocomposites onto the bacterial cell surface, which caused the cell layer to be damaged and intracellular ingredients to be discharged; (2) development of Zn/Ti ions and their electrostatic interaction with the bacterial cell membrane; (3) formation and accumulation of reactive oxygen species, with oxidative potential that caused damage DNA and proteins of microbes. Titanium oxide can cause loss of membrane integrity by oxidizing phospholipids using hydroxyl radicals and causing membrane fluidity and loss of cellular contents, and eventually microbial cell lysis [37,38]. All these factors could cumulatively initiate the cell death phenomenon in bacterial and fungal cultures in our experimental analyses. A recent report that revealed the antibacterial activity of bio-nanocomposites made utilizing garlic extract, which is employed in commercial applications such as food preservation, supports our findings [39].  Furthermore, we have tested the anticancer efficacy of nanocomposites using a MOLT4 blood cancer cell line, an in vitro experimental model. Treatment for leukemia is highly challenging due to the development of LSCs. LSCs have been found to participate in the progression of drug resistance in leukemic patients, rendering the patients and drug researchers in challenging conditions for reducing mortality rate [40]. MOLT4 cells treated using escin bio-nanocomposites showed significant cytotoxicity and loss of morphological architecture along with a substantial diminution in the total number of MOLT4 cells in culture plates. This denotes the escin bio-nanocomposites were capable of inducing cytotoxicity probably via the generation of ROS and pro-oxidants to trigger oxidative stress following penetration into the cells. Our observations demonstrated that the addition of Escin bio-nanocomposites in MOLT4 cells for 24 h induced a higher number of apoptotic cells, as shown in AO/EtBr staining, and increased the expression of ROS and loss of MMP. The study indicating that TiO 2 colloidal nanocomposite alone is capable of triggering cell death in leukemia cell line U937 in vitro is in agreement with our findings [41]. It is also possible that ZnO-TiO 2 -chitosan-escin bio-nanocomposites escalated oxidative stress while contrarily blocking NF-kB cascade. Many anticancer drugs confer drug resistance to cancer cells by activating NF-kB and activating anti-apoptotic pathways [42]. Interestingly, nanocomplexes using selenium have been shown to inhibit nF-kB p65 in hepatic inflammatory conditions [43]. In the near future, this could be the possible aspect to examine the antitumor effect of escin bio-nanocomposites generated in our study.

Conclusion
This study used a green synthesis method to create a ZnO-TiO 2 -chitosan-escin bio-nanocomposites. Following that, we investigated their anticancer potential by inhibiting cell growth and promoting apoptosis in MOLT4 cells. The bio-nanocomposites modified mitochondrial membrane potential and increased ROS generation in vitro. In addition, treatment with bio-nanocomposites increased the activation of apoptotic cell death. Further, it also showed antimicrobial effects on the growth of different pathogenic species in our in vitro experimental models. Hence, we recommended escin ZnO-TiO 2 -chitosan complex bio-nanocomposites for further evaluation in drug testing studies and designing new drugs using various plant phytochemicals. The findings confirmed that bio-nanocomposites of ZnO-TiO 2 -chitosan-escin could be potentially used to treat various leukemia and boost immune system functions. Data availability statement: The corresponding author will provide the data upon reasonable request.