Titanium dioxide nanoparticles treated by UVC and IR radiation-induced positive changes in a cancer cell line treatment

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F. Saad, Yasser A. Selim, Mona Hamada, Mohammed Elywa This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8373569/v1 This work is licensed under a CC BY 4.0 License Status: Under Revision Version 1 posted 14 You are reading this latest preprint version Abstract The aim of this study is to describe the effect of TiO₂ nanoparticles (NPs) synthesized with green tea extract, as a natural product on cancer cell lines, for example, the human hepatocellular cancer cell line, and their interaction with phototherapies such as ultraviolet (UV) and infrared (IR) therapy for anticancer treatment. TiO₂ (NPs) was synthesized in a procedure of green tea as one of the most antioxidant plant-derived natural products. Titanium dioxide nanoparticles (TiO₂ NPs) samples were exposed to UVC and IR radiation. The qualitative impacts of these treatments have been analyzed by means of ultraviolet/visible (UV/Vis) spectrophotometry in the range 200–800 nm, considering optical properties, as well as the cytotoxic effect, which was evaluated by measuring the numbers of viable cells, which were determined by the MTT test. The use of TiO₂ in conjunction with ultraviolet or infrared radiation has shown a remarkable ability to enhance the killing of cancer cells by generating reactive oxygen species. Therefore, the TiO₂ group and the activating treatment showed great potential and could be an effective therapeutic strategy against cancer. The final treatment results proved to be highly dependent on the specific order of the UV dose and had a lesser effect in the case of IR radiation. The results clearly showed that the value for the IC50 of cell viability of the HuH-7 cell line, which equals 36.40 ± 7.80 µg/ml, at 4 h UVC exposure was significantly lower than the IC 50 values for the other UVC exposure interval times. In addition, the exposure at the same dose had a better effect on normal cells (WI-38 cell line) and provided a greater value for this IC 50 parameter compared to both samples of control and standardized chemotherapy, whereas the IR treatment did not have a similar trend. The results also demonstrate that the production of TiO₂ nanoparticles by green synthesis with biological extracts is an effective technique to produce non-toxic materials for cancer treatment. Despite the good results achieved from using nanotechnology in medicinal treatment, it is essential that clinical applications remain limited, requiring additional studies on live animals that resemble the human body. TIO2 nanoparticles Photocatalysts. UVC-irradiation IR- irradiation cytotoxic effects Cancer cell line treatment anticancer activity Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 Figure 10 1. Introduction In the point of view of bio-medical science, healthy cells were negatively impacted by cancer therapy. One essential cancer treatment method had been radiotherapy. The development of radiation oncology will depend on the ongoing integration of scientific discoveries from various disciplines. Nanoscience in medicine was a new field of study with great potential to influence radiation oncology. Nanoscale materials had a number of special qualities that make them ideal for used in radiation oncology, including improved permeability and an efficient retention effect. Particle therapy, also known as cancer radiation therapy with energetic charged particles, was a novel therapeutic approach that offers significant advantages over traditional radiotherapy due to the unique ballistic characteristics and increased biological efficacy of energetic ions [ 1 ]. The main drawbacks of radiation therapy for cancer were as follows: first, the possibility of damage to the surrounding healthy tissues, and the other one, that some tumor cells might receive lower radiation intensity because they are located further from the radiation source. Even low-energy photons, such as UVC radiation, which have an approximate kinetic energy ranging from 5 to 10 electron volts, can exhibit nearly similar behavior. High-energy ionizing photon radiation, such as gamma rays or X-rays, was mostly used for ionizing biological components and/or water. In some rare cases, cancer tissues were targeted with particulate radiation, such as beta or alpha particles, or electron, proton, or neutron beams. [ 2 , 3 ]. Titanium dioxide (TiO2), also known as titania, is a member of the transition metal oxides family and can be found in a variety of crystalline forms due to its high photocatalytic activity, low cost, low toxicity, excellent chemical and thermal stability, and safety for both humans and the environment. When the crystalline phases were formed, three titanium (IV) ions shared six oxygen ions, resulting in the formula TiO6/3, which is equivalent to TiO2. The arrangement and distortion of these octahedral units created a distinctive crystallization pattern where each polymorph grows in three dimensions. The shapes and structures of the three polymorphs differ significantly from each other. TiO2 can be prepared using a variety of techniques, including thermal methods, hydrothermal methods, sol-gel methods, chemical vapor deposition, and electrodeposition. However, these techniques require a lot of energy as well as hazardous materials and solvents. 'Green synthesis,' a recent development in nanotechnology, uses biological systems such as microbes and plants [ 5 ]. Chinese camellia leaves were used to make green tea. They were originally domesticated in East Asia, and this plant can grow to the size of a shrub or a tree. Chinese camellia plants are currently cultivated in countries in Asia, the Middle East, and Africa. In addition, green tea has long been used to improve digestion, regulate blood sugar and body temperature, reduce gas, and enhance mental performance [ 6 ]. The strong presence of antioxidants known as polyphenols was the main factor responsible for the health benefits of green tea. In fact, polyphenols appear to be more effective antioxidants than vitamin C [ 7 ]. Small metallic nanoparticles have been proposed as potential nanomedicines to enhance radiation performance, as briefly discussed above. This approach, which aims to increase the cytotoxic effect on tumor cells while preserving healthy tissue, seeks to enrich tumors with nanoparticles to intensify the effects of radiation in the tumor. This appears to be a critical challenge in radiotherapy. However, the radio-resistance of certain malignant tumors is the main reason for the ineffectiveness of radiotherapy. [ 8 ]. It has also been found that titanium dioxide is useful in killing cancer cells through photocatalytic chemistry, and its mechanism of action includes generating reactive oxygen species when photoactivated by UVC rays [ 9 ]. In the present investigation, we propose to synthesis TiO 2 (NPs) nanoparticles of green tea as one of the most antioxidant planta natural products for radiotherapy with a focus on its enhancement in vitro anti-tumor activities and mainly to investigate the impact of UVC and IR radiation on these Titanium nanoparticles as radiation responses against the malignant cells. 2. Materials and Methods 2.1. Green synthesis of TiO 2 NPs The standard process for creating TiO 2 .NPs was dissolving green tea extract in 150 mL of boiling distilled water. Green tea extract was then mixed with 0.01 mol of titanium tetraisopropoxide (TTIP) for five minutes at 70°C while being stirred. After the pH of the solution was adjusted to basic (pH 9.0), neutral (pH 7.0), or acidic (pH 5.0) by gradually adding ammonium hydroxide (NH4 (OH)) solution while continuously stirring for 30 min., a yellow solution with white precipitates was generated. To extract TiO 2 (NPs), the precipitate was centrifuged at 8500 rpm for 10 min., then repeatedly cleaned with distilled water and dried at room temperature. [ 7 , 10 ]. In the current study, the main component of the extract is **polyphenolic compounds**. These phenols primarily act as capping agents, enhancing the stability of the synthesized nanoparticles. By adhering to the surfaces of the nanoparticles, the phenols prevent aggregation and increase the stability of the dispersion. Therefore, in the present system, the extract functions as a **stabilizer** for the nanoparticles, not as a reducing agent. This role is well-supported in the scientific literature, which demonstrates the strong effects of polyphenolic extracts in capping and stabilization during nanoparticle preparation. [ 11 ]. 2.2. Absorbance spectral analysis of TiO 2 NPs 2.2.1. UV–irradiation TiO2 (NPs) samples were exposed to UVC ultraviolet radiation for different exposure times ranging from 0.5 to 10 hours, using a UVC lamp emitting at a wavelength of 254 nm (Brand: Philips-Netherlands, Order Code: 500–00-820913283, Origin: Poland). The UVC lamp has an electrical power of 11 W. This lamp was placed 1 cm from the studied sample. During the exposure, and after each hour, the UVC lamp was turned off for 5 minutes to prevent excessive internal heating of the sample. 2.2.2. Infrared Radiation Exposure Infrared radiation exposure was carried out using a 250 W IR lamp, positioned 1 cm from the output source. The total exposure times for TiO2 (NPs) samples to infrared radiation ranged from 2.5 to 20 minutes. The integrated IR exposure times of the TiO 2 (NPs) ranged from 2.5 to 20 min. 2.3. In vitro toxicity measurements: Evaluation of cytotoxic effects of certain chemical compound 2.3.1. Mammalian cell lines WI 38 (Normal human lung fibroblast cells) and HuH-7cells (human Hepatocellular cancer cell line) were obtained from the American Type Culture Collection (ATCC, Rockville, MD). 2.3.2. Chemicals used Dimethyl sulfoxide (DMSO), Fetal Bovine serum, MTT and trypan blue dye was purchased from Sigma (St. Louis, Mo., USA). RPMI-1640, DMEM, HEPES buffer solution, L-glutamine, gentamycin and 0.25% Trypsin-EDTA were purchased from Lonza (Belgium). 2.3.3. Cell line Propagation: For WI 38 cells, cell growth was stimulated in Dulbecco's Modified Eagle Medium (DMEM) supplemented with 10% heat-inactivated fetal bovine serum, 1% L-glutamine, HEPES buffer, and 50 µg/ml gentamicin. For HuH-7 cells, cells were grown in RPMI-1640 medium supplemented with 10% heat-inactivated fetal calf serum and 50 µg/ml gentamicin. All cells were then maintained at 37°C in a humidified atmosphere containing 5% carbon dioxide, and they were partially cultured twice a week. 2.3.4. Cytotoxicity evaluation using viability assay In the cytotoxicity test, 100 µl of growth media was used to seed cells in a 96 well plate at a concentration of 1×104 cells/well. After 24 h of seeding, fresh medium with varying quantities of the test sample was introduced. Using a multichannel pipette, confluent cell monolayers were distributed into 96 well, flat bottomed microtiter plates (Falcon, NJ, USA) together with serial two fold dilutions of the investigated chemical compound. For 24 h, the microtiter plates were kept in a humidified incubator with five percent CO 2 at 37 o C. For every test sample concentration, three wells were utilized. Control cells were either treated with or without DMSO and without the test sample. It was discovered that the experiment was unaffected by the small amount of DMSO (maximum 0.1%) in the well. In Corning 96 well tissue culture plates, the tumor cell lines were suspended in media at a concentration of 5x104 cells/well for antitumor tests. The plates were then incubated for a full day. In order to obtain twelve concentrations for each drug, the tested compounds were thereafter put to 96 well plates (three duplicates). For every 96 well plate, six vehicle controls with media or 0.5% DMSO were run as a control. A colorimetric approach was used to determine the viable cell yield after the cells were incubated. The MTT test was used to count the number of viable cells after a 24-hour incubation period. In short, 100 µl of new culture RPMI 1640 medium without phenol red and 10 µl of the 12 mM MTT stock solution (5 mg of MTT in 1 mL of PBS) were added to each well, including the untreated controls, after the media was taken out of the 96-well plates. After that, the 96 well plates were incubated for four hours at 37°C with 5% CO 2 . After removing an 85 µl aliquot of the media from each well, 50 µl of DMSO was added, thoroughly mixed with a pipette, and incubated for 10 minutes at 37°C. The number of viable cells was then determined by measuring the optical density at 590 nm using a microplate reader (Sun Rise, TECAN, Inc., USA). The percentage of viability was then computed as [(ODt/ODc)] x 100%, where ODt is the mean optical density of wells treated with the tested sample and ODC is the mean optical density of untreated cells. Using Graph Pad Prism software, graphic plots of the dose response curve for each concentration were used to estimate the IC 50 , which is the concentration that inhibits 50% of tumor cell viability, and CC 50 , which refers to normal cells. (San Diego, CA. USA) [ 12 , 13 ]. 2.3.5. Microscopic observation of the tumor cells treated with the compounds As previously mentioned in conducting an anti-tumor activity, this experiment was carried out. The plates were inverted to remove the medium after treating the experimental concentration, then the wells were washed three times with 300 microliters of phosphate-buffered saline (pH 7.2). Next, the cells were fixed on the plate for 15 minutes at room temperature using 10% formalin. The fixed cells were then stained with 100 microliters of 0.25% crystal violet for 20 minutes. The stain was removed, and the plates were rinsed with deionized water to remove excess stain and then left to dry. Images were captured showing morphological changes compared to control cells using an inverted microscope (CKX41; Olympus, Japan) equipped with a digital microscope camera at 100x magnification. [ 14 ]. 3. Characterization methods Titanium dioxide (NPs) was examined using ultraviolet-visible spectroscopy, transmission electron microscopy (TEM), scanning electron microscopy (SEM), and zeta potential to study metals, semiconductors, and insulators in their bulk, suspended, thin film, and nanostructured forms. Optical absorption spectroscopy is a very useful method. Some insulating and semiconducting materials have an optical energy gap. When the photon energy is insufficient to excite electrons from the valence level to the conduction level, absorption does not occur. A UV-Vis spectrophotometer (Spectro UV–Vis 2800, USA) was used to measure transmittance and absorption within the wavelength range of 200–800 nm to record the optical absorption spectrum. Eq. 1 was used to calculate the optical band gap energy of TiO2 particles. [ 15 ]. E = h c /⅄ (1) Where h is Planck constant = 6.626x 10 − 34 Joule sec, C is Velocity of light = 2.99 x 10 ^8 meter/sec and ⅄ is absorption peak value. TEM and SEM microscopes are useful tools for investigating the shape, size, composition, and even chemical composition of solids depending on the type of microscope used. Some very powerful microscopes have the ability to distinguish structures at the atomic level. TEM is an efficient and high-precision tool that can perform analysis at the nanoscale. TEM is used to measure the size of nanoparticles, grains, and small crystals, the arrangement of atoms in matter, as well as the formation of new phases in very small amounts, which the XRD meter cannot explain. The analysis was performed using a JEOL electron microscope (JEM-100CX) and images were taken at different magnifications (100 nm). The morphology and imaging of the composite nanoparticles were examined using a scanning electron microscope (SEM). A Zeiss LEO Supra 55VP Field Emission and SEM Zeiss 1530 were used to capture SEM images. For sample preparation, the composite nanoparticle suspensions were diluted tenfold in their dispersing medium before being placed directly onto a polished aluminum sample holder. The samples were dried under vacuum. Afterwards, the samples were coated with gold using a sputter coater (EMITECH K450X) [ 16 ]. The "particle charge" in a liquid medium is measured by the zeta potential, sometimes known as the electrokinetic potential. The most common format for the results is in millivolts. When particles come into contact with a liquid, a zeta potential is generated. The surface charge of a particle is the result of the interaction between the functional groups on its surface and the surrounding liquid. What is known as the electrical double layer (EDL) is formed when a concentration of oppositely charged ions is attracted to this charge. The combination of the primary surface charge and the charge of the accumulated layer is known as the zeta potential. The stability of the dispersion is described in terms of the zeta potential. Typically, a low zeta potential indicates that the particles are unstable and may aggregate, coagulate, or clump together. On the other hand, a high zeta potential suggests potential stability. However, the zeta potential is influenced by several variables, such as pH, buffer solution concentration, temperature, and ionic strength. The zeta potential was measured using a Zeta Sizer analyzer from Malvern PANalytical Ltd, model number NS500. [ 17 ]. 4. Statistical Analysis Data are presented as mean and the standard deviation (SD) for each studied group. Statistical significance between experimental groups and the control was calculated using the independent two-sample T-test (Welch’s t-test), which accounts for potential differences in variances between groups [ 18 ]. A p-value, less than 0.05, was set at less than 0.05 and showed a statistical significance for all comparisons. All analyses were conducted using standard statistical software according to standard criteria. 4. Results and Discussion 4.1. Study of UV-Visible spectroscopy The Fig. 1 (a, b) represents the UV-Vis spectrum of pristine and UVC and IR irradiated TiO 2 (NPs) samples. The variation of the optical band-gap energy, by means of the Eq. (1), versus UV and IR exposure time intervals for the TiO 2 (NPs) samples exposed to different time intervals of UVC and IR radiation compared to the unexposed (control) sample are shown in Fig. 1 (c, d). In the case of UV exposure, the optical band-gap energy generally exhibits a parabola curve behavior. Thus, the optical band-gap energies show a gradual fairly decrease up to 4 h which corresponds to the minimum value of the band gap energy, followed by a significant increase thereafter. As will discuss later in the text, this minimum value of optical band gap energy plays a key role showing that the UV-irradiated TIO 2 (NPs) exhibited high treatment capacity and promoted the disappearance of the malignant cells relative to the control sample. In the case of IR-exposure, UV/Visible measurements have shown that the energy band gap decreases with increasing exposure time. So, a red shift towards the higher wavelengths is seen as the IR-exposure time increased [ 19 ]. This is caused by a rise in interatomic spacing ripening temperature [ 20 ]. The resulting energy band gap values are lower at increasing exposure time compared to the 3.2 eV energy gap for pure anatase, 3.0 for pure rutile form, and the TiO 2 nano powder mixed-phase results for gas sensing applications [ 20 – 22 ]. The interaction of both UVC and IR radiation with malignant molecules is different, where IR radiation as a thermal radiation causes molecules to undergo vibrational transitions, whereas UVC as a high energy UV photon (6.1–4.3 eV) causes many biological molecules to undergo electronic transitions. This makes the response of these molecules against UVC and IR radiation clearly differentiated, and this significantly affects their efficiency in destroying these malignant cells. 4.2. TEM analysis TEM analysis can be used to understand the crystalline characteristics and size of TiO 2 (NPs) before and after irradiation as shown in Fig. 2 (a, b, c). The size of TiO 2 (NPs) before irradiation (control) range from 4.16, 7.86, 10.8, 12.5, 18.8, 24.7, 30.6, 20.4, 16.5, 37, 28.8, 14 nm as shown in Fig. 2 (a). In addition, the size of TiO 2 (NPs) after UV exposure at 4 h, showed 4.97, 23.3, 21.7, 16.9, 36.3, 26.5, 14.9 nm, this result slightly near to the size of the control, this confirm that the UV-irradiation does not affect the size of TIO 2 (NPS). Whereas the Fig. 2 (c) showed aggregation of the particles after IR-exposure at 20 min compare to the shape of particles in Fig. 2 (a, b). 4.3. HR-SEM analysis A concentrated beam of high-energy electrons is used by the SEM to produce a range of signals at the surface of specimens. The signals that originate from electron- sample interactions provide information about the sample including external morphology (texture), chemical composition, and crystalline structure and orientation of materials making up the sample. Figure 3 shows TiO 2 NPs were uniformly distributed and cylindrical in shape. 4.4. Zeta potential analysis Zeta potential is used to measure the stability of colloidal suspensions of particles depends on the charged species present at the particles surface that impair the aggregation and the consequent collapse. A High absolute zeta potential value indicates a high electric charge on the surface of the NPs. It describes strong repellent forces among the particles, preventing aggregation and stabilizing NPs in the buffer solution. In natural conditions (pH close to 7.2), the values of zeta potential were extremely negative (-15.6 ± 0.043) mV as seen in Fig. 4 . The agglomeration observed in the TEM images for the treated samples is likely a result of the photocatalytic activity of the TiO 2 nanoparticles. Upon UV and IR irradiation, Surface functionalization with stabilizing agents: We will investigate surface functionalization of the TiO 2 nanoparticles with stabilizing agents such as polyethylene glycol (PEG) or chitosan. PEGylation, for example, creates a steric hindrance layer around the nanoparticles, physically preventing them from aggregating even if their zeta potential is reduced. This approach is widely used to enhance the stability and biocompatibility of nanoparticles in biological media. Optimizing Suspension Medium: The nanoparticles were suspended in a medium that may not have been fully optimized for long-term colloidal stability under irradiation. We will explore the use of different suspending media or the addition of dispersants to the current medium to maintain a high degree of dispersion throughout the treatment process. 4.5. Cytotoxic assay When TiO 2 is exposed to ultraviolet photons, thus, electrons move from their ground state in the valence band to an excited state in the conduction band, creating positive holes in the valence band. Following this occurrence, molecules that have been adsorbed onto the catalyst's surface, like water and oxygen, are capable of undergoing redox reactions [ 23 ]. While reduction happens at the conduction band with the help of increased photoelectrons, oxidation occurs at the valence band via electron acceptors [ 24 ]. So, it is known that exposing TiO 2 NPs to a constituents-hour UV exposure causes a significant number of bridging hydroxyl species with a strong acidic character to form. (pKa 2.9). On the other hand, terminal hydroxyl species (Ti-OH) are more alcaline (pKa = 12.7) and their existence in physiological fluids encourages a more neutral surface charge. Given that the former species, which are more thermodynamically stable, require that water molecules replace oxygen atoms that are expelled during the h + -driven oxidation of O 2 − anions, we may hypothesize that the brief irradiation duration utilized in this and previous works [ 25 ], may favours the later species. The less negative surface charge seen for all materials could be caused by an abundance of terminal hydroxyl species after brief irradiation. Reactive oxygen species that are produced after irradiation can spread from the nanoparticles and cause cell death in nearby tissues. As a result, numerous TiO 2 nano- and micro particle uses in photodynamic treatment are being investigated. TiO 2 nanoparticles can also be used as drug carriers, delivering medications to diseased body regions while sparing healthy tissues from injury [ 26 ]. The relation between surviving cells and drug concentration is plotted to get the survival curve of each tumor cell line after treatment with the specified compound.as shown in Figs. 5 (a) and 6 (a). Cancer, a malignant condition caused by uncontrolled cell growth, is thought to be the second greatest cause of death in the world. The hepatocellular cancers are the most dangerous and are linked to the highest mortality rates among the various cancer types [ 27 ]. Creating novel anticancer medications with fewer side effects and more selectivity and efficacy is one of the most difficult fields in contemporary scientific research [ 28 ]. The cytotoxicity of the tested materials (TIO 2 .NPs) was investigated before and after irradiation. From the MTT assay results present in Fig. 6 (a) and supplementary Table 1. It is interesting to note that the exposure of TIO 2 to 4 h from UV lamp showed a profound selective cytotoxic effect on the hepatocellular cancer cell lines with appreciable lower cytotoxic activity on the normal WI38 cells. IC 50 of HuH-7 cells at UV-irradiation were 12.8, 60.43 and 36.4 µg/mL by the 5-FU Standard, the control (TIO 2 .NPs before irradiation) and TIO 2 .NPs after 4 h UV-irradiation respectively. While IC 50 of normal WI38 cells at UV-irradiation were 65.1, 64.7 and 110.8 µg/mL by the 5-FU Standard, the control (TIO 2 .NPs before irradiation) and TIO 2 .NPs after 4 h UV-irradiation respectively. Limited information for HuH-7 cells exposed to UVC: Although the cytotoxicity of TiO 2 nanoparticles has been thoroughly studied, precise IC 50 values for how they affect HuH-7 cells exposed to UVC are not publicly accessible [ 29 ]. The majority of published research on TiO 2 and cancer cells either uses different light sources (such as UVA or visible light) or concentrates on other cell lines. A TiO 2 /thiopolyurethane composite's limited IC 50 value was discovered in a study conducted on a different hepatocellular carcinoma cell line (HepG2) [ 30 ]. Our findings about the notable cytotoxic effect of TiO 2 nanoparticles on HuH-7 cells following UVC exposure are in line with the established mechanisms of TiO 2 photocatalysis and photodynamic therapy, according to the body of existing literature [ 31 – 33 ]. The cytotoxicity's dose-dependent character and UV's vital role From lowest to greatest energy level, UV radiation can be further separated into UV-C (200–290 nm), UV-B (290–320 nm), and UV-A (320–400 nm). Since UV-C radiation cannot get through the Earth's atmosphere, UV-B and UV-A radiation exposures are the principal causes of photo biological effects in humans [ 34 , 35 ]. According to [ 28 , 35 ], both kind of UV radiations induce photo biological consequences such as sunburn, pigmentation issues, immunosuppression, cancer, and damage to DNA and other cellular components [ 35 – 37 ]. So, this result of morphology images, showed that at low dose of UV-irradiation until reach to 4 h of UV-irradiation showed an effect on the tumor cells and less effect on the normal cell as shown in Fig. 7 , (a), but in the case of increasing the UV-irradiation dose, IC 50 of HuH-7 cells at UV-irradiation were 270.7 and 81.2 µg/mL TIO 2 .NPs after 8 h, 10 h of UV-irradiation respectively. And also, IC 50 of normal WI38 cells at UV-irradiation were 84.3 and 230.9 µg/mL respectively as shown in Table 3 . This means that at increasing the UV-irradiation the energy increase and then after certain time, it can cause injure in the cells. From the morphology images, as shown in Figs. 8 and 9 , we note that the 5-FU sample showed an image free of infected cells compared to the HuH-7cells, and then the control sample, which is the sample of titanium nanoparticles that are not irradiated, a clear image appeared and was free of infected cells, but to a lesser extent than the 5-FU stander. This is because chemotherapy gives a better result, but it has its drawbacks, which is that after a period of time, it can cause death, so we resorted to radiation therapy, which does not cause injury to normal cells, and through it, the affected cells can be eliminated with a result closer to the result of chemotherapy, through an image Morphology of a sample irradiated with 4 hours of UV-irradiation, the image appeared at the highest concentration, free of infected cells, and the result was closer to the image of the 5-Fu stander and better than that of the control sample. When evaluating the cytotoxic activity of titanium dioxide nanoparticles (TiO₂ NPs) against HuH-7 and WI-38 cell lines, statistical analysis using T-tests revealed important differences in response to various treatments. The obtained p-values were instrumental in determining the statistical significance of the cytotoxic effects at different incubation times. Specifically, IC₅₀ values at several time points such as 0.5h, 1h, and 4h, showed p-values less than 0.05 in both cell lines, indicating statistically significant cytotoxic effects as compared to the control group as shown in Table 3 . This result confirms that exposure to TiO₂ (NPs) under these conditions elicits meaningful cytotoxicity, while other time points with p-values greater than 0.05 suggest a non-significant impact [ 18 ]. Our findings highlight the importance of integrating rigorous statistical approaches for validating cytotoxicity outcomes, as previously emphasized by contemporary studies and recommendations in the literature. Table 3 Statistical validation (e.g., p-values, confidence intervals) for differences in IC₅₀ values of Titanium dioxide nanoparticle (TIO 2 NP S ) effect on cell viability of HuH-7 cell line and WI-38 cell line after exposure to UV-radiation. Samples HuH-7 cell line WI-38 cell line P Value Confidence intervals (95%) P Value Confidence intervals (95%) 0.5 h 0.047 [61.98, 107.32] 0.013 [67.75, 108.49] 1 h 0.046 [66.07, 103.24] 0.040 [74.87, 101,37] 2 h 0.890 [64.99, 104.31] 0.160 [70.86, 105.38] 3 h 0.780 [64.45, 104.85] 0.350 [68.74,107.50] 4 h 0.020 [56.81, 109.49] 0.026 [76.39, 99.85] 6 h 0.990 [66.41, 102.89] 0.800 [72.62, 103.62] 8 h 0.100 [75.55, 93.75] 0.350 [74.67, 101.57] 10 h 0.540 [68.00, 101.30] 0.0013 [79.99, 96.25] Effects mediated by IR were once thought to just be heat-related. While IR is considering as thermal radiation and give the same effect of annealing temperature, the average size of synthetic TiO 2 NPs grew larger as the annealing temperature was raised. Annealing temperatures gives the transformation of phases from anatase to rutile. Due to the high surface energy of the nanoparticles, an aggregation of synthetic TiO 2 nanoparticles occurred during annealing temperatures. [ 21 , 38 ]. In the present study, the TiO 2 NPs effect on cell viability of WI-38 cell line, the cell viability of TiO 2 NPs after IR-irradiation remains constant until reach to concentration 62.5 µg/ml then it became decreased in all samples. However, exposure to 10 and 20 min remain constant until reach to concentration 31.25 µg/ml, the viability (%) is 87.29 ± 1.02 and 92.19 ± 1.95 decrease to 60.13 ± 1.11 and 71.31 ± 2.14 at concentration 1000 µg/m respectively, as seen in Fig. 6 (b). While on HuH-7cells, the cell viability of TiO 2 NPs before and after IR-irradiation remain constant until reach to concentration 62.5 µg/ml. while in case of exposure to 10 and 20 min remain constant until reach to concentration 31.25 µg/ml, the viability (%) is 86.19 ± 0.92 and 91.19 ± 1.02 decrease to 62.11 ± 3.11 and 74.33 ± 2.16 at concentration 1000 µg/m respectively. as seen in Fig. 5 (b). The values of IC 50 in the case of HuH-7cells and WI38 cells exposed to IR irradiation increased with increasing exposure time. IC 50 of HuH-7cells is 12.8 ± 3.2, 60.43 ± 7.9 and 80.10 ± 21.30 µg/mL for the 5-FU Standard, the control and TIO 2 .NPs after 20 min IR-irradiation respectively, as shown on Fig. 7 (b) and supplementary Table 2. While IC 50 of normal WI38 cells showed 65.10 ± 9.18, 64.70 ± 12.84 and 98.9o ± 20.2o µg/mL for the 5-FU Standard, the control and TIO 2 .NPs after 20 min IR-irradiation respectively. This means that IR -irradiation makes heat to the particles and effect on the poly phenolic acid and make aggregation to the molecules so caused injured to the cell. It should be noted that the statistical variations of minimum, maximum, mean, median, range and standard deviation values of titanium dioxide nanoparticle TiO 2 (NPs) effect on cell viability of HuH-7 cell line and WI-38 cell line before and after exposure to UVC and IR radiation, respectively. It is necessary to understand some of the limitations and advantages of this approach in the cancer cell line treatment as it needs more study and research to reach better results before clinic stage. Cell lines from either people or non-human animals are used in vitro investigations, where they are combined with the novel drug being developed in a Petri dish or test tube. In vitro research has various advantages. Firstly, the most evident advantage is that they do not have the negative effects of animal testing because they do not damage the person or animal from which the cell cultures were created. Secondly, in vitro approaches have the advantages of being relatively inexpensive to set up and maintain, as well as being dependable, effective, and producing solid results. In contrast to in vitro investigations, in vivo studies are carried out inside a living creature. This occurs in animal test subjects during preclinical studies. In vivo experiments can involve either human or animal subjects in clinical trials, the main drawback of in vitro research can be overcome by in vivo investigations since they can show how a drug affects the body as whole rather than just isolated cells. This makes it possible for in vivo studies to more clearly see potential interactions, which can help them forecast a drug's safety, toxicity, and effectiveness. This aids scientists in predicting how potential medications will affect human disease. In vivo investigations have their own significant drawbacks despite the fact that they address the problem with in vitro studies. Significant ethical issues surround in vivo research, particularly for preclinical investigations where only animal models are allowed. In future, it is hopeful that we can apply this approach to inject these TIO 2 (NPs) synthesized in a typical procedure of green tea as one of the most antioxidant planta natural products in laboratory mice or larger animals to be activated directly by each of the photons of UVC or IR radiation which confirms the validity of the approach used. TiO 2 nanoparticles were selected from all the data because of their photocatalytic properties, which include: Nanoparticles are well-known Photocatalysts in terms of their mechanism of action. This indicates that they have the ability to absorb light energy, particularly in the ultraviolet spectrum, and use it to produce extremely reactive oxygen species (ROS), like hydroxyl radicals (•OH). Therapeutic uses, their application in photodynamic therapy (PDT) are based on this characteristic. The researchers can cause localized oxidative stress by activating the nanoparticles with a light source (in this case, UVC), which causes lipid peroxidation, protein oxidation, and DNA damage inside the cancer cells. This is a focused strategy to cause the cancerous cells to undergo apoptosis, or programmed cell death. TiO 2 in combination with UVC and nanoparticles are anticipated to create a strong and quick cytotoxic [ 39 ]. The ideal band gap (Eg) of our manufactured nanomaterial, which showed the maximum efficacy, is the "minimum value" that we refer to in our study. This value has physical significance since it controls how well the material absorbs light energy. The material may absorb a wider range of light, including visible light, when its band gap is less (within an ideal range). As a result, when exposed to photo irradiation, more electron-hole pairs (e−/h+) are produced. The formation of Reactive Oxygen Species (ROS) such hydroxyl radicals (⋅OH) and superoxide anions (⋅O₂⁻) is directly boosted by the higher concentration of charge carriers (electrons and holes) brought about by the smaller band gap. In photocatalysis, these species are principally in charge of breaking down organic contaminants due to their strong oxidizing potential. Thus, the increased photocatalytic activity seen in our studies can be explained by the direct correlation between the lowest band gap value we measured and the highest rate of ROS formation [ 40 ]. The increased cytotoxic effects are explained by the same mechanism. When used in biomedical settings, the effectively produced ROS target essential cellular components in addition to contaminants. These organisms cause extreme oxidative stress in cells, which damages proteins, DNA, and the cell membrane and ultimately results in programmed cell death (apoptosis). This indicates that the strong cytotoxic effects we saw are directly related to the high ROS production efficiency made possible by the ideal minimum band gap [ 41 ]. One important element influencing our TiO 2 nanoparticles' reliable and consistent cytotoxic effects is their size stability. A trustworthy dose-response relationship depends on the surface area-to-volume ratio and photocatalytic activity remaining consistent during the experiment, which is ensured by this stability. The reactive surface area of nanoparticles may decrease as they aggregate or undergo size changes, which might lower the production of Reactive Oxygen Species (ROS) and, consequently, their cytotoxic effectiveness. Consequently, the persistent cytotoxic effects seen in our MTT assay are strongly supported by the stability of our nanoparticles' size [ 42 ]. According to certain research, the in vitro toxicity of nanoparticles can be significantly reduced as they aggregate over time. The significance of regulating physicochemical parameters for reliable and efficient therapeutic applications is thus supported by our findings, which show a steady particle size [ 43 ]. This thorough review article offers a great summary of how agglomeration affects TiO 2 nanoparticle toxicity. It is a great resource to utilize when comparing your results to those of other studies because it addresses findings from a variety of studies. The research highlights that one of the main causes of the diversity in published nanotoxicity data is variations in size and aggregation state. Although our results imply that ROS may be the mediator of the observed cytotoxicity, it is crucial to remember that this preliminary investigation did not include direct measurement of ROS levels (for example, using the DCFDA assay). To conclusively validate and measure the role of ROS generation in the mechanistic pathway, more research will be necessary. Our synthetic compound's noteworthy [e.g., cytotoxic, antibacterial, and anti-inflammatory] properties make it a viable option for a number of potential futures uses: As a Lead Compound for Therapeutics: This study provides evidence of the molecular scaffold's medicinal potential. The development of a new class of [e.g., anti-bacterial, anti-cancer] drugs may result from future research concentrating on medicinal physics and chemistry efforts to optimize the compound's structure to increase potency, improve its pharmacokinetic profile, and decrease off-target toxicity. As a tool for basic research: The compound can be used as a chemical probe to study the specific biological pathways it modulates. Understanding its mechanism of action could uncover new therapeutic targets or reveal novel aspects of disease pathology. In combination therapy: Future investigations could explore the synergistic effects of our compound when combined with existing standard drugs. This could potentially lead to more effective treatment regimens that reduce drug resistance and lower required dosages." As a Basic Research Tool: To investigate the particular biological pathways that the substance affects, it can be employed as a chemical probe. Knowing how it works may help identify new therapeutic targets or new facets of the pathophysiology of the disease. Future research could examine our compound's synergistic benefits in combination therapy with currently available conventional medications. This may result in more efficient treatment plans that cut dosage requirements and lessen drug resistance. 5. Conclusion We have introduced a new approach of TIO 2 nanoparticles treated by UVC and IR radiation, in vitro, on the growth of HuH-7 cells (human Hepatocellular cancer cell line), to obtain a unique technique for anticancer activity over exposure periods of 0.5–10 h of UVC radiation and from 2.5–20 min of IR radiation, respectively. The obtained results suggest that the extent of UVC-induced TIO 2 nanoparticles’ activation is dose-dependent, whereas in the case of IR treatment it shows non similar trend. Our results imply that this new technique enhances TIO 2 nanoparticles efficiency treated by UVC radiation, allowing high-degree treatment of cancer cells. UVC treatment has proven to be the most efficient process in the current study, which induces continuous steady decreases in the values of IC 50 of HuH-7 cells, and a highest increase in the values of WI-38 cell line. The decrease in the optical band-gap energy as a result of UVC exposure at 4 h interval period can be mainly ascribed to the formation of unsaturated bonds, in which π-π * transitions of delocalized electrons can occur. The high absorption of UVC light by the molecules and atoms of malignant cells, particularly in the far-UVC region, is attributed to absorption by peptide bonds of proteins and lipids, and which form an inverted resonance peak. To our knowledge, the cytotoxic effects of TIO 2 nanoparticles treated by UVC, in vitro, on the growth of HuH-7 cells (human Hepatocellular cancer cell line has never been reported before. The optical band gap energy measurements clearly confirm that the positive changes brought to the malignant cells as a result of UVC exposure are physico-biological in nature, especially when minimize the values of these optical band gap energies. Finally, we concluded that the chemotherapy gives relatively better result than our used method, but it has its drawbacks, which is that after a period of time, it can cause death, so we resorted to UVC radiation therapy, which may cause less harm to normal cells. This finding agrees well with the published data in the literature on both chemotherapy and radiotherapy which also support the powerful accuracy of the method used. Declarations Competing interests The authors declare no competing interests. Ethical approval Not applicable. Consent to participate Not applicable. Consent to publish Not applicable. Author Contribution A.F. Saad: Conceptualization, Methodology, Writing - Reviewing, Editing; Yasser A. Selim: Conceptualization, Methodology, Writing - Reviewing; Mona Hamada: Data curation, Investigation, Formal analysis, Writing-original draft; Mohammed Elywa: Co-supervision, Investigation Data Availability All data generated or analyzed during this study are included in this published article. The datasets generated during and/or analyzed during the current study are available from the corresponding author on reasonable request. References Yu M, Zhiying S, Johnny V, Orit K, Andrew Z (2016) W Cancer Nanatechnol. ; 7–11 Hogle WP (2006) The state of the art in radiation therapy. Semin Oncol Nurs 22:212–220 Mallick I, Waldron JN (2009) Radiation therapy for head and neck cancers. 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Supplementary Files SupplementaryTable1.docx SupplementaryTable2.docx Cite Share Download PDF Status: Under Revision Version 1 posted Editorial decision: Revision requested 11 May, 2026 Reviews received at journal 05 Mar, 2026 Reviews received at journal 02 Mar, 2026 Reviewers agreed at journal 26 Feb, 2026 Reviewers agreed at journal 24 Feb, 2026 Reviewers agreed at journal 23 Feb, 2026 Reviewers agreed at journal 23 Feb, 2026 Reviewers agreed at journal 23 Feb, 2026 Reviews received at journal 14 Jan, 2026 Reviewers agreed at journal 14 Jan, 2026 Reviewers invited by journal 09 Jan, 2026 Editor assigned by journal 29 Dec, 2025 Submission checks completed at journal 28 Dec, 2025 First submitted to journal 28 Dec, 2025 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. 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1","display":"","copyAsset":false,"role":"figure","size":72350,"visible":true,"origin":"","legend":"\u003cp\u003ea), b) Absorbance data from a TiO\u003csub\u003e2\u003c/sub\u003e (NPs) exposed to UV-IR radiation for exposure time ranging From 0.5 to 10 h and 2.5 to 20 min respectively ,(c),\u0026nbsp; (d) The integrated absorbance versus UV-IR-exposure time.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-8373569/v1/b38fcf7a949fdf7f7bf3749a.png"},{"id":100395399,"identity":"0e7a6ce2-b850-4ef0-b199-e3a83a622bd8","added_by":"auto","created_at":"2026-01-16 11:38:54","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":34409,"visible":true,"origin":"","legend":"\u003cp\u003eVariation of the optical band gap energy of TiO\u003csub\u003e2\u003c/sub\u003e (NPs exposed to UV-IR at different Exposure time ranging from 0.5 to 10 h and 2.5 to 20 min respectively.\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-8373569/v1/0b252513e7a8de33372b4306.png"},{"id":100396097,"identity":"6f336bf3-5caf-4ce4-97d2-05c77746a6eb","added_by":"auto","created_at":"2026-01-16 11:39:55","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":544761,"visible":true,"origin":"","legend":"\u003cp\u003eTEM micrograph of TiO\u003csub\u003e2\u003c/sub\u003e (NPs), (a) pre – UV and IR exposure (control),\u0026nbsp; (b) post-UV exposure at 4 h. (c) post-IR exposure at 20 min.\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-8373569/v1/de41cb308bdeffb1047b77a2.png"},{"id":100395397,"identity":"73c500b2-67d7-4c35-bdc6-f490df8c1a9e","added_by":"auto","created_at":"2026-01-16 11:38:54","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":295384,"visible":true,"origin":"","legend":"\u003cp\u003eSEM images of synthesized TiO2 (NPs), (a) Magnification 15.000 x at 5µm, (b) Magnification 30.000 x at 3µm, (c) Magnification 60.000 x at 1µm.\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-8373569/v1/bf15e25acee6b24b65184e10.png"},{"id":100395414,"identity":"f270b401-768e-4a75-b514-9c7957d5479a","added_by":"auto","created_at":"2026-01-16 11:39:01","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":66182,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eZeta potential of green synthesis TIO\u003c/strong\u003e\u003csub\u003e\u003cstrong\u003e2\u003c/strong\u003e\u003c/sub\u003e\u003cstrong\u003e (NPs).\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-8373569/v1/655c27fa4bd65c35fb9414b6.png"},{"id":100395516,"identity":"6d7f1cd2-2545-4959-9514-d6528bb48277","added_by":"auto","created_at":"2026-01-16 11:39:05","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":79769,"visible":true,"origin":"","legend":"\u003cp\u003ea), TiO\u003csub\u003e2\u003c/sub\u003e (NPs) pre UV exposure (control) and post UV exposure at different time of radiation from 0.5 to 10 h. b), TiO\u003csub\u003e2\u003c/sub\u003e (NPs) pre IR exposure (control) and post IR exposure at different time of radiation from 2.5 to 20 min respectively, effect on cell viability at HuH-7cells.\u003c/p\u003e","description":"","filename":"6.png","url":"https://assets-eu.researchsquare.com/files/rs-8373569/v1/ac651d468f0932bd56ad6772.png"},{"id":100395977,"identity":"b3814e4c-ab02-4979-bb6c-9d85aea4f621","added_by":"auto","created_at":"2026-01-16 11:39:42","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":96498,"visible":true,"origin":"","legend":"\u003cp\u003ea),\u003cstrong\u003e \u003c/strong\u003eTiO\u003csub\u003e2\u003c/sub\u003e (NPs) pre UV exposure (control) and post UV exposure at different time of radiation from 0.5 to 10 h. b)\u003cstrong\u003e \u003c/strong\u003eTiO\u003csub\u003e2\u003c/sub\u003e (NPs) pre IR exposure (control) and post IR exposure at different time of radiation from\u0026nbsp; 2.5 to 20min respectively, effect on cell viability at WI-38 cell line.\u003c/p\u003e","description":"","filename":"7.png","url":"https://assets-eu.researchsquare.com/files/rs-8373569/v1/e261078f5b175355ee29dc76.png"},{"id":100396049,"identity":"31a7b12f-653a-4e33-bff2-b6b131741547","added_by":"auto","created_at":"2026-01-16 11:39:51","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":32390,"visible":true,"origin":"","legend":"\u003cp\u003ea) Relation between IC\u003csub\u003e50\u003c/sub\u003e and TIO\u003csub\u003e2\u003c/sub\u003e (NPs) exposed to UVC radiation at different exposure time, b) Relation between IC\u003csub\u003e50\u003c/sub\u003e and TIO\u003csub\u003e2\u003c/sub\u003e (NPs) exposed to IR radiation at different exposure time, for the HuH-7 cells and WI-38 cells.\u003c/p\u003e","description":"","filename":"8.png","url":"https://assets-eu.researchsquare.com/files/rs-8373569/v1/8a1b02f12b79a78f79d0a786.png"},{"id":100395572,"identity":"8042bae4-3625-4392-a8dd-30df0587d15e","added_by":"auto","created_at":"2026-01-16 11:39:08","extension":"png","order_by":9,"title":"Figure 9","display":"","copyAsset":false,"role":"figure","size":632111,"visible":true,"origin":"","legend":"\u003cp\u003eMorphological characterization of Hepatocellular cancer cell line for non-treated and treated cell at different concentration after 24h treatment: (a) Stands for non-treated cell, (b) Cell treated with 5-FU stander at different concentration; (b1) 500 µg/ml, (b2) 100 µg/ml, (b3) 20 µg/ml, (b4) 4 µg/ml (c) TiO2NPs before exposure to UV- radiation (control), at different concentration; (c1) 500 µg/ml, (c2) 100 µg/ml, (c3) 20 µg/ml, (c4) 4 µg/ml (d) Figure Click here to access/download;Figure;Figure Captions.docx TiO2NPs Post exposure to UV - radiation at 3h, at different concentration; (d1) 500 µg/ml, (d2) 100 µg/ml, (d3) 20 µg/ml, (d4) 4 µg/ml. (e) TiO2 NPs Post exposure to UV - radiation at 4h, at different concentration; (e1) 500 µg/ml, (e2) 100 µg/ml, (e3) 20 µg/ml, (e4) 4 µg/ml. (f) TiO2NPs Post exposure to UV - radiation at 6h, at different concentration; (f1) 500 µg/ml, (f2) 100 µg/ml, (f3) 20 µg/ml, (f4) 4 µg/ml (g) TiO2NPs Post exposure to UV - radiation at 8h, at different concentration; (g1) 500 µg/ml, (g2) 100 µg/ml, (g3) 20 µg/ml, (g4) 4 µg/ml. (h) TiO2NPs Post exposure to UV - radiation at 10h. at different concentration; (h1) 500 µg/ml, (h2) 100 µg/ml, (h3) 20 µg/ml, (h4) 4 µg/ml.\u003c/p\u003e","description":"","filename":"9.png","url":"https://assets-eu.researchsquare.com/files/rs-8373569/v1/036012657dfd2d6456e68c75.png"},{"id":100395952,"identity":"aed2623a-ea00-439e-9a87-5dc225974066","added_by":"auto","created_at":"2026-01-16 11:39:38","extension":"png","order_by":10,"title":"Figure 10","display":"","copyAsset":false,"role":"figure","size":327684,"visible":true,"origin":"","legend":"\u003cp\u003eMorphological characterization of Hepatocellular cancer cell line for non-treated and treated cell with Vinblastine sulfate reference sample and TiO2NPs after exposure to UV- radiation at 4h at different concentration after 24h treatment: (a) Stands for nontreated cell, (b) Cell treated with 5-FU stander at different concentration; (b1) 500 µg/ml, (b2) 100 µg/ml, (b3) 20 µg/ml, (b4) 4 µg/ml.(e) TiO2NPs post exposure to UV- radiation at 4 h, at different concentration; (e1) 500 µg/ml, (e2) 100 µg/ml, (e3) 20 µg/ml, (e4) 4 µg/ml.\u003c/p\u003e","description":"","filename":"10.png","url":"https://assets-eu.researchsquare.com/files/rs-8373569/v1/296cb36fe56a24aa266e582d.png"},{"id":100413786,"identity":"b5916b54-7fee-4a6f-b75a-f3d47cbd97ec","added_by":"auto","created_at":"2026-01-16 13:18:09","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2922816,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8373569/v1/cf2d6835-130c-40d4-9f15-054639bc2b2e.pdf"},{"id":100395679,"identity":"5823246b-7e51-4f55-aa79-4eeb405aa7f8","added_by":"auto","created_at":"2026-01-16 11:39:19","extension":"docx","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":19237,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryTable1.docx","url":"https://assets-eu.researchsquare.com/files/rs-8373569/v1/de25afb65a32fbee54cd3a1b.docx"},{"id":100395324,"identity":"dbe2f417-f1e6-497e-afcc-afd61d9d1a00","added_by":"auto","created_at":"2026-01-16 11:38:50","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":16056,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryTable2.docx","url":"https://assets-eu.researchsquare.com/files/rs-8373569/v1/a33f6ecf415f6d625442322c.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Titanium dioxide nanoparticles treated by UVC and IR radiation-induced positive changes in a cancer cell line treatment","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eIn the point of view of bio-medical science, healthy cells were negatively impacted by cancer therapy. One essential cancer treatment method had been radiotherapy. The development of radiation oncology will depend on the ongoing integration of scientific discoveries from various disciplines. Nanoscience in medicine was a new field of study with great potential to influence radiation oncology. Nanoscale materials had a number of special qualities that make them ideal for used in radiation oncology, including improved permeability and an efficient retention effect. Particle therapy, also known as cancer radiation therapy with energetic charged particles, was a novel therapeutic approach that offers significant advantages over traditional radiotherapy due to the unique ballistic characteristics and increased biological efficacy of energetic ions [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe main drawbacks of radiation therapy for cancer were as follows: first, the possibility of damage to the surrounding healthy tissues, and the other one, that some tumor cells might receive lower radiation intensity because they are located further from the radiation source. Even low-energy photons, such as UVC radiation, which have an approximate kinetic energy ranging from 5 to 10 electron volts, can exhibit nearly similar behavior. High-energy ionizing photon radiation, such as gamma rays or X-rays, was mostly used for ionizing biological components and/or water. In some rare cases, cancer tissues were targeted with particulate radiation, such as beta or alpha particles, or electron, proton, or neutron beams. [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eTitanium dioxide (TiO2), also known as titania, is a member of the transition metal oxides family and can be found in a variety of crystalline forms due to its high photocatalytic activity, low cost, low toxicity, excellent chemical and thermal stability, and safety for both humans and the environment. When the crystalline phases were formed, three titanium (IV) ions shared six oxygen ions, resulting in the formula TiO6/3, which is equivalent to TiO2. The arrangement and distortion of these octahedral units created a distinctive crystallization pattern where each polymorph grows in three dimensions. The shapes and structures of the three polymorphs differ significantly from each other. TiO2 can be prepared using a variety of techniques, including thermal methods, hydrothermal methods, sol-gel methods, chemical vapor deposition, and electrodeposition. However, these techniques require a lot of energy as well as hazardous materials and solvents. 'Green synthesis,' a recent development in nanotechnology, uses biological systems such as microbes and plants [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. Chinese camellia leaves were used to make green tea. They were originally domesticated in East Asia, and this plant can grow to the size of a shrub or a tree. Chinese camellia plants are currently cultivated in countries in Asia, the Middle East, and Africa. In addition, green tea has long been used to improve digestion, regulate blood sugar and body temperature, reduce gas, and enhance mental performance [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. The strong presence of antioxidants known as polyphenols was the main factor responsible for the health benefits of green tea. In fact, polyphenols appear to be more effective antioxidants than vitamin C [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. Small metallic nanoparticles have been proposed as potential nanomedicines to enhance radiation performance, as briefly discussed above. This approach, which aims to increase the cytotoxic effect on tumor cells while preserving healthy tissue, seeks to enrich tumors with nanoparticles to intensify the effects of radiation in the tumor. This appears to be a critical challenge in radiotherapy. However, the radio-resistance of certain malignant tumors is the main reason for the ineffectiveness of radiotherapy. [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. It has also been found that titanium dioxide is useful in killing cancer cells through photocatalytic chemistry, and its mechanism of action includes generating reactive oxygen species when photoactivated by UVC rays [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. In the present investigation, we propose to synthesis TiO\u003csub\u003e2\u003c/sub\u003e (NPs) nanoparticles of green tea as one of the most antioxidant planta natural products for radiotherapy with a focus on its enhancement in vitro anti-tumor activities and mainly to investigate the impact of UVC and IR radiation on these Titanium nanoparticles as radiation responses against the malignant cells.\u003c/p\u003e \u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003cdiv id=\"Sec4\" class=\"Section3\"\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section3\"\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003cdiv id=\"Sec7\" class=\"Section3\"\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section3\"\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section3\"\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section3\"\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section3\"\u003e \u003c/div\u003e \u003c/div\u003e"},{"header":"2. Materials and Methods","content":"\u003ch2\u003e2.1. Green synthesis of TiO\u003csub\u003e2\u003c/sub\u003e NPs\u003c/h2\u003e\u003cp\u003eThe standard process for creating TiO\u003csub\u003e2\u003c/sub\u003e.NPs was dissolving green tea extract in 150 mL of boiling distilled water. Green tea extract was then mixed with 0.01 mol of titanium tetraisopropoxide (TTIP) for five minutes at 70°C while being stirred. After the pH of the solution was adjusted to basic (pH 9.0), neutral (pH 7.0), or acidic (pH 5.0) by gradually adding ammonium hydroxide (NH4 (OH)) solution while continuously stirring for 30 min., a yellow solution with white precipitates was generated. To extract TiO\u003csub\u003e2\u003c/sub\u003e (NPs), the precipitate was centrifuged at 8500 rpm for 10 min., then repeatedly cleaned with distilled water and dried at room temperature. [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. In the current study, the main component of the extract is **polyphenolic compounds**. These phenols primarily act as capping agents, enhancing the stability of the synthesized nanoparticles. By adhering to the surfaces of the nanoparticles, the phenols prevent aggregation and increase the stability of the dispersion. Therefore, in the present system, the extract functions as a **stabilizer** for the nanoparticles, not as a reducing agent. This role is well-supported in the scientific literature, which demonstrates the strong effects of polyphenolic extracts in capping and stabilization during nanoparticle preparation. [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e].\u003c/p\u003e\u003ch2\u003e2.2. Absorbance spectral analysis of TiO\u003csub\u003e2\u003c/sub\u003eNPs\u003c/h2\u003e\u003ch2\u003e2.2.1. UV–irradiation\u003c/h2\u003e\u003cp\u003eTiO2 (NPs) samples were exposed to UVC ultraviolet radiation for different exposure times ranging from 0.5 to 10 hours, using a UVC lamp emitting at a wavelength of 254 nm (Brand: Philips-Netherlands, Order Code: 500–00-820913283, Origin: Poland). The UVC lamp has an electrical power of 11 W. This lamp was placed 1 cm from the studied sample. During the exposure, and after each hour, the UVC lamp was turned off for 5 minutes to prevent excessive internal heating of the sample.\u003c/p\u003e\u003ch2\u003e2.2.2. Infrared Radiation Exposure\u003c/h2\u003e\u003cp\u003eInfrared radiation exposure was carried out using a 250 W IR lamp, positioned 1 cm from the output source. The total exposure times for TiO2 (NPs) samples to infrared radiation ranged from 2.5 to 20 minutes. The integrated IR exposure times of the TiO\u003csub\u003e2\u003c/sub\u003e (NPs) ranged from 2.5 to 20 min.\u003c/p\u003e\u003ch2\u003e2.3. In vitro toxicity measurements: Evaluation of cytotoxic effects of certain chemical compound\u003c/h2\u003e\u003ch2\u003e2.3.1. Mammalian cell lines\u003c/h2\u003e\u003cp\u003eWI 38 (Normal human lung fibroblast cells) and HuH-7cells (human Hepatocellular cancer cell line) were obtained from the American Type Culture Collection (ATCC, Rockville, MD).\u003c/p\u003e\u003ch2\u003e2.3.2. Chemicals used\u003c/h2\u003e\u003cp\u003eDimethyl sulfoxide (DMSO), Fetal Bovine serum, MTT and trypan blue dye was purchased from Sigma (St. Louis, Mo., USA). RPMI-1640, DMEM, HEPES buffer solution, L-glutamine, gentamycin and 0.25% Trypsin-EDTA were purchased from Lonza (Belgium).\u003c/p\u003e\u003ch2\u003e2.3.3. Cell line Propagation:\u003c/h2\u003e\u003cp\u003eFor WI 38 cells, cell growth was stimulated in Dulbecco's Modified Eagle Medium (DMEM) supplemented with 10% heat-inactivated fetal bovine serum, 1% L-glutamine, HEPES buffer, and 50 µg/ml gentamicin. For HuH-7 cells, cells were grown in RPMI-1640 medium supplemented with 10% heat-inactivated fetal calf serum and 50 µg/ml gentamicin. All cells were then maintained at 37°C in a humidified atmosphere containing 5% carbon dioxide, and they were partially cultured twice a week.\u003c/p\u003e\u003ch2\u003e2.3.4. Cytotoxicity evaluation using viability assay\u003c/h2\u003e\u003cp\u003eIn the cytotoxicity test, 100 µl of growth media was used to seed cells in a 96 well plate at a concentration of 1×104 cells/well. After 24 h of seeding, fresh medium with varying quantities of the test sample was introduced. Using a multichannel pipette, confluent cell monolayers were distributed into 96 well, flat bottomed microtiter plates (Falcon, NJ, USA) together with serial two fold dilutions of the investigated chemical compound. For 24 h, the microtiter plates were kept in a humidified incubator with five percent CO\u003csub\u003e2\u003c/sub\u003e at 37 \u003csup\u003eo\u003c/sup\u003eC. For every test sample concentration, three wells were utilized. Control cells were either treated with or without DMSO and without the test sample. It was discovered that the experiment was unaffected by the small amount of DMSO (maximum 0.1%) in the well. In Corning 96 well tissue culture plates, the tumor cell lines were suspended in media at a concentration of 5x104 cells/well for antitumor tests. The plates were then incubated for a full day. In order to obtain twelve concentrations for each drug, the tested compounds were thereafter put to 96 well plates (three duplicates). For every 96 well plate, six vehicle controls with media or 0.5% DMSO were run as a control. A colorimetric approach was used to determine the viable cell yield after the cells were incubated. The MTT test was used to count the number of viable cells after a 24-hour incubation period. In short, 100 µl of new culture RPMI 1640 medium without phenol red and 10 µl of the 12 mM MTT stock solution (5 mg of MTT in 1 mL of PBS) were added to each well, including the untreated controls, after the media was taken out of the 96-well plates. After that, the 96 well plates were incubated for four hours at 37°C with 5% CO\u003csub\u003e2\u003c/sub\u003e. After removing an 85 µl aliquot of the media from each well, 50 µl of DMSO was added, thoroughly mixed with a pipette, and incubated for 10 minutes at 37°C. The number of viable cells was then determined by measuring the optical density at 590 nm using a microplate reader (Sun Rise, TECAN, Inc., USA). The percentage of viability was then computed as [(ODt/ODc)] x 100%, where ODt is the mean optical density of wells treated with the tested sample and ODC is the mean optical density of untreated cells. Using Graph Pad Prism software, graphic plots of the dose response curve for each concentration were used to estimate the IC\u003csub\u003e50\u003c/sub\u003e, which is the concentration that inhibits 50% of tumor cell viability, and CC\u003csub\u003e50\u003c/sub\u003e, which refers to normal cells. (San Diego, CA. USA) [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e].\u003c/p\u003e\u003ch2\u003e2.3.5. Microscopic observation of the tumor cells treated with the compounds\u003c/h2\u003e\u003cp\u003eAs previously mentioned in conducting an anti-tumor activity, this experiment was carried out. The plates were inverted to remove the medium after treating the experimental concentration, then the wells were washed three times with 300 microliters of phosphate-buffered saline (pH 7.2). Next, the cells were fixed on the plate for 15 minutes at room temperature using 10% formalin. The fixed cells were then stained with 100 microliters of 0.25% crystal violet for 20 minutes. The stain was removed, and the plates were rinsed with deionized water to remove excess stain and then left to dry. Images were captured showing morphological changes compared to control cells using an inverted microscope (CKX41; Olympus, Japan) equipped with a digital microscope camera at 100x magnification. [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e].\u003c/p\u003e"},{"header":"3. Characterization methods","content":"\u003cp\u003eTitanium dioxide (NPs) was examined using ultraviolet-visible spectroscopy, transmission electron microscopy (TEM), scanning electron microscopy (SEM), and zeta potential to study metals, semiconductors, and insulators in their bulk, suspended, thin film, and nanostructured forms. Optical absorption spectroscopy is a very useful method. Some insulating and semiconducting materials have an optical energy gap. When the photon energy is insufficient to excite electrons from the valence level to the conduction level, absorption does not occur. A UV-Vis spectrophotometer (Spectro UV\u0026ndash;Vis 2800, USA) was used to measure transmittance and absorption within the wavelength range of 200\u0026ndash;800 nm to record the optical absorption spectrum. Eq.\u0026nbsp;1 was used to calculate the optical band gap energy of TiO2 particles. [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eE\u0026thinsp;=\u0026thinsp;h c /⅄ (1)\u003c/p\u003e \u003cp\u003eWhere h is Planck constant\u0026thinsp;=\u0026thinsp;6.626x 10 \u003csup\u003e\u0026minus;\u0026thinsp;34\u003c/sup\u003e Joule sec, C is Velocity of light\u0026thinsp;=\u0026thinsp;2.99 x 10 ^8 meter/sec and ⅄ is absorption peak value. TEM and SEM microscopes are useful tools for investigating the shape, size, composition, and even chemical composition of solids depending on the type of microscope used. Some very powerful microscopes have the ability to distinguish structures at the atomic level. TEM is an efficient and high-precision tool that can perform analysis at the nanoscale. TEM is used to measure the size of nanoparticles, grains, and small crystals, the arrangement of atoms in matter, as well as the formation of new phases in very small amounts, which the XRD meter cannot explain. The analysis was performed using a JEOL electron microscope (JEM-100CX) and images were taken at different magnifications (100 nm). The morphology and imaging of the composite nanoparticles were examined using a scanning electron microscope (SEM). A Zeiss LEO Supra 55VP Field Emission and SEM Zeiss 1530 were used to capture SEM images. For sample preparation, the composite nanoparticle suspensions were diluted tenfold in their dispersing medium before being placed directly onto a polished aluminum sample holder. The samples were dried under vacuum. Afterwards, the samples were coated with gold using a sputter coater (EMITECH K450X) [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. The \"particle charge\" in a liquid medium is measured by the zeta potential, sometimes known as the electrokinetic potential. The most common format for the results is in millivolts. When particles come into contact with a liquid, a zeta potential is generated. The surface charge of a particle is the result of the interaction between the functional groups on its surface and the surrounding liquid. What is known as the electrical double layer (EDL) is formed when a concentration of oppositely charged ions is attracted to this charge. The combination of the primary surface charge and the charge of the accumulated layer is known as the zeta potential. The stability of the dispersion is described in terms of the zeta potential. Typically, a low zeta potential indicates that the particles are unstable and may aggregate, coagulate, or clump together. On the other hand, a high zeta potential suggests potential stability. However, the zeta potential is influenced by several variables, such as pH, buffer solution concentration, temperature, and ionic strength. The zeta potential was measured using a Zeta Sizer analyzer from Malvern PANalytical Ltd, model number NS500. [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e].\u003c/p\u003e"},{"header":"4. Statistical Analysis","content":"\u003cp\u003eData are presented as mean and the standard deviation (SD) for each studied group. Statistical significance between experimental groups and the control was calculated using the independent two-sample T-test (Welch\u0026rsquo;s t-test), which accounts for potential differences in variances between groups [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. A p-value, less than 0.05, was set at less than 0.05 and showed a statistical significance for all comparisons. All analyses were conducted using standard statistical software according to standard criteria.\u003c/p\u003e"},{"header":"4. Results and Discussion","content":"\u003cdiv id=\"Sec15\" class=\"Section2\"\u003e\n \u003ch2\u003e4.1. Study of UV-Visible spectroscopy\u003c/h2\u003e\n \u003cp\u003eThe Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e (a, b) represents the UV-Vis spectrum of pristine and UVC and IR irradiated TiO\u003csub\u003e2\u003c/sub\u003e (NPs) samples. The variation of the optical band-gap energy, by means of the Eq.\u0026nbsp;(1), versus UV and IR exposure time intervals for the TiO\u003csub\u003e2\u003c/sub\u003e (NPs) samples exposed to different time intervals of UVC and IR radiation compared to the unexposed (control) sample are shown in Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e (c, d). In the case of UV exposure, the optical band-gap energy generally exhibits a parabola curve behavior. Thus, the optical band-gap energies show a gradual fairly decrease up to 4 h which corresponds to the minimum value of the band gap energy, followed by a significant increase thereafter. As will discuss later in the text, this minimum value of optical band gap energy plays a key role showing that the UV-irradiated TIO\u003csub\u003e2\u003c/sub\u003e (NPs) exhibited high treatment capacity and promoted the disappearance of the malignant cells relative to the control sample. In the case of IR-exposure, UV/Visible measurements have shown that the energy band gap decreases with increasing exposure time. So, a red shift towards the higher wavelengths is seen as the IR-exposure time increased [\u003cspan class=\"CitationRef\"\u003e19\u003c/span\u003e]. This is caused by a rise in interatomic spacing ripening temperature [\u003cspan class=\"CitationRef\"\u003e20\u003c/span\u003e]. The resulting energy band gap values are lower at increasing exposure time compared to the 3.2 eV energy gap for pure anatase, 3.0 for pure rutile form, and the TiO\u003csub\u003e2\u003c/sub\u003e nano powder mixed-phase results for gas sensing applications [\u003cspan class=\"CitationRef\"\u003e20\u003c/span\u003e\u0026ndash;\u003cspan class=\"CitationRef\"\u003e22\u003c/span\u003e]. The interaction of both UVC and IR radiation with malignant molecules is different, where IR radiation as a thermal radiation causes molecules to undergo vibrational transitions, whereas UVC as a high energy UV photon (6.1\u0026ndash;4.3 eV) causes many biological molecules to undergo electronic transitions. This makes the response of these molecules against UVC and IR radiation clearly differentiated, and this significantly affects their efficiency in destroying these malignant cells.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec16\" class=\"Section2\"\u003e\n \u003ch2\u003e4.2. TEM analysis\u003c/h2\u003e\n \u003cp\u003eTEM analysis can be used to understand the crystalline characteristics and size of TiO\u003csub\u003e2\u003c/sub\u003e (NPs) before and after irradiation as shown in Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e (a, b, c). The size of TiO\u003csub\u003e2\u003c/sub\u003e (NPs) before irradiation (control) range from 4.16, 7.86, 10.8, 12.5, 18.8, 24.7, 30.6, 20.4, 16.5, 37, 28.8, 14 nm as shown in Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e (a). In addition, the size of TiO\u003csub\u003e2\u003c/sub\u003e (NPs) after UV exposure at 4 h, showed 4.97, 23.3, 21.7, 16.9, 36.3, 26.5, 14.9 nm, this result slightly near to the size of the control, this confirm that the UV-irradiation does not affect the size of TIO\u003csub\u003e2\u003c/sub\u003e (NPS). Whereas the Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e (c) showed aggregation of the particles after IR-exposure at 20 min compare to the shape of particles in Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e (a, b).\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec17\" class=\"Section2\"\u003e\n \u003ch2\u003e4.3. HR-SEM analysis\u003c/h2\u003e\n \u003cp\u003eA concentrated beam of high-energy electrons is used by the SEM to produce a range of signals at the surface of specimens. The signals that originate from electron- sample interactions provide information about the sample including external morphology (texture), chemical composition, and crystalline structure and orientation of materials making up the sample. Figure\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003e shows TiO\u003csub\u003e2\u003c/sub\u003eNPs were uniformly distributed and cylindrical in shape.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec18\" class=\"Section2\"\u003e\n \u003ch2\u003e\u003cstrong\u003e4.4. Zeta potential analysis\u003c/strong\u003e\u003c/h2\u003e\n \u003cp\u003eZeta potential is used to measure the stability of colloidal suspensions of particles depends on the charged species present at the particles surface that impair the aggregation and the consequent collapse. A High absolute zeta potential value indicates a high electric charge on the surface of the NPs. It describes strong repellent forces among the particles, preventing aggregation and stabilizing NPs in the buffer solution. In natural conditions (pH close to 7.2), the values of zeta potential were extremely negative (-15.6\u0026thinsp;\u0026plusmn;\u0026thinsp;0.043) mV as seen in Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003e.\u003c/p\u003e\n \u003cp\u003eThe agglomeration observed in the TEM images for the treated samples is likely a result of the photocatalytic activity of the TiO\u003csub\u003e2\u003c/sub\u003e nanoparticles. Upon UV and IR irradiation, Surface functionalization with stabilizing agents: We will investigate surface functionalization of the TiO\u003csub\u003e2\u003c/sub\u003e nanoparticles with stabilizing agents such as polyethylene glycol (PEG) or chitosan. PEGylation, for example, creates a steric hindrance layer around the nanoparticles, physically preventing them from aggregating even if their zeta potential is reduced. This approach is widely used to enhance the stability and biocompatibility of nanoparticles in biological media. Optimizing Suspension Medium: The nanoparticles were suspended in a medium that may not have been fully optimized for long-term colloidal stability under irradiation. We will explore the use of different suspending media or the addition of dispersants to the current medium to maintain a high degree of dispersion throughout the treatment process.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec19\" class=\"Section2\"\u003e\n \u003ch2\u003e4.5. Cytotoxic assay\u003c/h2\u003e\n \u003cp\u003eWhen TiO\u003csub\u003e2\u003c/sub\u003e is exposed to ultraviolet photons, thus, electrons move from their ground state in the valence band to an excited state in the conduction band, creating positive holes in the valence band. Following this occurrence, molecules that have been adsorbed onto the catalyst\u0026apos;s surface, like water and oxygen, are capable of undergoing redox reactions [\u003cspan class=\"CitationRef\"\u003e23\u003c/span\u003e]. While reduction happens at the conduction band with the help of increased photoelectrons, oxidation occurs at the valence band via electron acceptors [\u003cspan class=\"CitationRef\"\u003e24\u003c/span\u003e]. So, it is known that exposing TiO\u003csub\u003e2\u003c/sub\u003e NPs to a constituents-hour UV exposure causes a significant number of bridging hydroxyl species with a strong acidic character to form. (pKa 2.9). On the other hand, terminal hydroxyl species (Ti-OH) are more alcaline (pKa\u0026thinsp;=\u0026thinsp;12.7) and their existence in physiological fluids encourages a more neutral surface charge. Given that the former species, which are more thermodynamically stable, require that water molecules replace oxygen atoms that are expelled during the h\u003csup\u003e+\u003c/sup\u003e-driven oxidation of O\u003csub\u003e2\u003c/sub\u003e\u003csup\u003e\u0026minus;\u003c/sup\u003e anions, we may hypothesize that the brief irradiation duration utilized in this and previous works [\u003cspan class=\"CitationRef\"\u003e25\u003c/span\u003e], may favours the later species. The less negative surface charge seen for all materials could be caused by an abundance of terminal hydroxyl species after brief irradiation. Reactive oxygen species that are produced after irradiation can spread from the nanoparticles and cause cell death in nearby tissues. As a result, numerous TiO\u003csub\u003e2\u003c/sub\u003e nano- and micro particle uses in photodynamic treatment are being investigated. TiO\u003csub\u003e2\u003c/sub\u003e nanoparticles can also be used as drug carriers, delivering medications to diseased body regions while sparing healthy tissues from injury [\u003cspan class=\"CitationRef\"\u003e26\u003c/span\u003e].\u003c/p\u003e\n \u003cp\u003eThe relation between surviving cells and drug concentration is plotted to get the survival curve of each tumor cell line after treatment with the specified compound.as shown in Figs.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003e (a) and 6 (a). Cancer, a malignant condition caused by uncontrolled cell growth, is thought to be the second greatest cause of death in the world. The hepatocellular cancers are the most dangerous and are linked to the highest mortality rates among the various cancer types [\u003cspan class=\"CitationRef\"\u003e27\u003c/span\u003e]. Creating novel anticancer medications with fewer side effects and more selectivity and efficacy is one of the most difficult fields in contemporary scientific research [\u003cspan class=\"CitationRef\"\u003e28\u003c/span\u003e]. The cytotoxicity of the tested materials (TIO\u003csub\u003e2\u003c/sub\u003e.NPs) was investigated before and after irradiation. From the MTT assay results present in Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003e (a) and supplementary Table\u0026nbsp;1. It is interesting to note that the exposure of TIO\u003csub\u003e2\u003c/sub\u003e to 4 h from UV lamp showed a profound selective cytotoxic effect on the hepatocellular cancer cell lines with appreciable lower cytotoxic activity on the normal WI38 cells.\u003c/p\u003e\n \u003cp\u003eIC\u003csub\u003e50\u003c/sub\u003e of HuH-7 cells at UV-irradiation were 12.8, 60.43 and 36.4 \u0026micro;g/mL by the 5-FU Standard, the control (TIO\u003csub\u003e2\u003c/sub\u003e.NPs before irradiation) and TIO\u003csub\u003e2\u003c/sub\u003e.NPs after 4 h UV-irradiation respectively. While IC\u003csub\u003e50\u003c/sub\u003e of normal WI38 cells at UV-irradiation were 65.1, 64.7 and 110.8 \u0026micro;g/mL by the 5-FU Standard, the control (TIO\u003csub\u003e2\u003c/sub\u003e.NPs before irradiation) and TIO\u003csub\u003e2\u003c/sub\u003e.NPs after 4 h UV-irradiation respectively. Limited information for HuH-7 cells exposed to UVC: Although the cytotoxicity of TiO\u003csub\u003e2\u003c/sub\u003e nanoparticles has been thoroughly studied, precise IC\u003csub\u003e50\u003c/sub\u003e values for how they affect HuH-7 cells exposed to UVC are not publicly accessible [\u003cspan class=\"CitationRef\"\u003e29\u003c/span\u003e]. The majority of published research on TiO\u003csub\u003e2\u003c/sub\u003e and cancer cells either uses different light sources (such as UVA or visible light) or concentrates on other cell lines. A TiO\u003csub\u003e2\u003c/sub\u003e/thiopolyurethane composite\u0026apos;s limited IC\u003csub\u003e50\u003c/sub\u003e value was discovered in a study conducted on a different hepatocellular carcinoma cell line (HepG2) [\u003cspan class=\"CitationRef\"\u003e30\u003c/span\u003e]. Our findings about the notable cytotoxic effect of TiO\u003csub\u003e2\u003c/sub\u003e nanoparticles on HuH-7 cells following UVC exposure are in line with the established mechanisms of TiO\u003csub\u003e2\u003c/sub\u003e photocatalysis and photodynamic therapy, according to the body of existing literature [\u003cspan class=\"CitationRef\"\u003e31\u003c/span\u003e\u0026ndash;\u003cspan class=\"CitationRef\"\u003e33\u003c/span\u003e]. The cytotoxicity\u0026apos;s dose-dependent character and UV\u0026apos;s vital role\u003c/p\u003e\n \u003cp\u003eFrom lowest to greatest energy level, UV radiation can be further separated into UV-C (200\u0026ndash;290 nm), UV-B (290\u0026ndash;320 nm), and UV-A (320\u0026ndash;400 nm). Since UV-C radiation cannot get through the Earth\u0026apos;s atmosphere, UV-B and UV-A radiation exposures are the principal causes of photo biological effects in humans [\u003cspan class=\"CitationRef\"\u003e34\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e35\u003c/span\u003e]. According to [\u003cspan class=\"CitationRef\"\u003e28\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e35\u003c/span\u003e], both kind of UV radiations induce photo biological consequences such as sunburn, pigmentation issues, immunosuppression, cancer, and damage to DNA and other cellular components [\u003cspan class=\"CitationRef\"\u003e35\u003c/span\u003e\u0026ndash;\u003cspan class=\"CitationRef\"\u003e37\u003c/span\u003e]. So, this result of morphology images, showed that at low dose of UV-irradiation until reach to 4 h of UV-irradiation showed an effect on the tumor cells and less effect on the normal cell as shown in Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e7\u003c/span\u003e, (a), but in the case of increasing the UV-irradiation dose, IC\u003csub\u003e50\u003c/sub\u003e of HuH-7 cells at UV-irradiation were 270.7 and 81.2 \u0026micro;g/mL TIO\u003csub\u003e2\u003c/sub\u003e.NPs after 8 h, 10 h of UV-irradiation respectively. And also, IC\u003csub\u003e50\u003c/sub\u003e of normal WI38 cells at UV-irradiation were 84.3 and 230.9 \u0026micro;g/mL respectively as shown in Table\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003e. This means that at increasing the UV-irradiation the energy increase and then after certain time, it can cause injure in the cells. From the morphology images, as shown in Figs.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e8\u003c/span\u003e and \u003cspan class=\"InternalRef\"\u003e9\u003c/span\u003e, we note that the 5-FU sample showed an image free of infected cells compared to the HuH-7cells, and then the control sample, which is the sample of titanium nanoparticles that are not irradiated, a clear image appeared and was free of infected cells, but to a lesser extent than the 5-FU stander. This is because chemotherapy gives a better result, but it has its drawbacks, which is that after a period of time, it can cause death, so we resorted to radiation therapy, which does not cause injury to normal cells, and through it, the affected cells can be eliminated with a result closer to the result of chemotherapy, through an image Morphology of a sample irradiated with 4 hours of UV-irradiation, the image appeared at the highest concentration, free of infected cells, and the result was closer to the image of the 5-Fu stander and better than that of the control sample. When evaluating the cytotoxic activity of titanium dioxide nanoparticles (TiO₂ NPs) against HuH-7 and WI-38 cell lines, statistical analysis using T-tests revealed important differences in response to various treatments. The obtained p-values were instrumental in determining the statistical significance of the cytotoxic effects at different incubation times. Specifically, IC₅₀ values at several time points such as 0.5h, 1h, and 4h, showed p-values less than 0.05 in both cell lines, indicating statistically significant cytotoxic effects as compared to the control group as shown in Table\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003e. This result confirms that exposure to TiO₂ (NPs) under these conditions elicits meaningful cytotoxicity, while other time points with p-values greater than 0.05 suggest a non-significant impact [\u003cspan class=\"CitationRef\"\u003e18\u003c/span\u003e]. Our findings highlight the importance of integrating rigorous statistical approaches for validating cytotoxicity outcomes, as previously emphasized by contemporary studies and recommendations in the literature.\u003c/p\u003e\n \u003cp\u003e\u003cbr\u003e\u003c/p\u003e\n \u003cdiv class=\"gridtable\"\u003e\n \u003cdiv align=\"left\" class=\"colspec\"\u003e\u003cbr\u003e\u003c/div\u003e\n \u003cdiv align=\"char\" class=\"colspec\"\u003e\u003cbr\u003e\u003c/div\u003e\n \u003cdiv align=\"char\" class=\"colspec\"\u003e\u003cbr\u003e\u003c/div\u003e\u0026nbsp;\u0026nbsp;\u003ctable id=\"Tab1\" border=\"1\"\u003e\n \u003ccaption language=\"En\"\u003e\n \u003cdiv class=\"CaptionNumber\"\u003eTable 3\u003c/div\u003e\n \u003cdiv class=\"CaptionContent\"\u003e\n \u003cp\u003eStatistical validation (e.g., p-values, confidence intervals) for differences in IC₅₀ values of Titanium dioxide nanoparticle (TIO\u003csub\u003e2\u003c/sub\u003e NP\u003csub\u003eS\u003c/sub\u003e) effect on cell viability of HuH-7 cell line and WI-38 cell line after exposure to UV-radiation.\u003c/p\u003e\n \u003c/div\u003e\n \u003c/caption\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003cth align=\"left\" rowspan=\"2\"\u003e\n \u003cp\u003eSamples\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\" colspan=\"5\"\u003e\n \u003cp\u003eHuH-7 cell line \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;WI-38 cell line\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003eP Value\u003c/strong\u003e\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\" style=\"width: 34.1112%;\"\u003e\n \u003cp\u003e\u003cstrong\u003eConfidence intervals (95%)\u003c/strong\u003e\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\" style=\"width: 1.4312%;\"\u003e\u0026nbsp;\u003c/th\u003e\n \u003cth align=\"left\" style=\"width: 9.66%;\"\u003e\n \u003cp\u003e\u003cstrong\u003eP Value\u003c/strong\u003e\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003eConfidence intervals (95%)\u003c/strong\u003e\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.5 h\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.047\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" style=\"width: 34.1112%;\"\u003e\n \u003cp\u003e[61.98, 107.32]\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" style=\"width: 1.4312%;\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"char\" style=\"width: 9.66%;\"\u003e\n \u003cp\u003e0.013\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e[67.75, 108.49]\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1 h\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.046\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" style=\"width: 34.1112%;\"\u003e\n \u003cp\u003e[66.07, 103.24]\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" style=\"width: 1.4312%;\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"char\" style=\"width: 9.66%;\"\u003e\n \u003cp\u003e0.040\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e[74.87, 101,37]\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2 h\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.890\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" style=\"width: 34.1112%;\"\u003e\n \u003cp\u003e[64.99, 104.31]\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" style=\"width: 1.4312%;\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"char\" style=\"width: 9.66%;\"\u003e\n \u003cp\u003e0.160\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e[70.86, 105.38]\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e3 h\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.780\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" style=\"width: 34.1112%;\"\u003e\n \u003cp\u003e[64.45, 104.85]\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" style=\"width: 1.4312%;\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"char\" style=\"width: 9.66%;\"\u003e\n \u003cp\u003e0.350\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e[68.74,107.50]\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e4 h\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.020\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" style=\"width: 34.1112%;\"\u003e\n \u003cp\u003e[56.81, 109.49]\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" style=\"width: 1.4312%;\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"char\" style=\"width: 9.66%;\"\u003e\n \u003cp\u003e0.026\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e[76.39, 99.85]\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e6 h\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.990\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" style=\"width: 34.1112%;\"\u003e\n \u003cp\u003e[66.41, 102.89]\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" style=\"width: 1.4312%;\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"char\" style=\"width: 9.66%;\"\u003e\n \u003cp\u003e0.800\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e[72.62, 103.62]\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e8 h\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.100\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" style=\"width: 34.1112%;\"\u003e\n \u003cp\u003e[75.55, 93.75]\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" style=\"width: 1.4312%;\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"char\" style=\"width: 9.66%;\"\u003e\n \u003cp\u003e0.350\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e[74.67, 101.57]\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e10 h\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.540\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" style=\"width: 34.1112%;\"\u003e\n \u003cp\u003e[68.00, 101.30]\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" style=\"width: 1.4312%;\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"char\" style=\"width: 9.66%;\"\u003e\n \u003cp\u003e0.0013\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e[79.99, 96.25]\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n \u003c/div\u003e\n \u003cp\u003eEffects mediated by IR were once thought to just be heat-related. While IR is considering as thermal radiation and give the same effect of annealing temperature, the average size of synthetic TiO\u003csub\u003e2\u003c/sub\u003eNPs grew larger as the annealing temperature was raised. Annealing temperatures gives the transformation of phases from anatase to rutile. Due to the high surface energy of the nanoparticles, an aggregation of synthetic TiO\u003csub\u003e2\u003c/sub\u003e nanoparticles occurred during annealing temperatures. [\u003cspan class=\"CitationRef\"\u003e21\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e38\u003c/span\u003e]. In the present study, the TiO\u003csub\u003e2\u003c/sub\u003e NPs effect on cell viability of WI-38 cell line, the cell viability of TiO\u003csub\u003e2\u003c/sub\u003e NPs after IR-irradiation remains constant until reach to concentration 62.5 \u0026micro;g/ml then it became decreased in all samples. However, exposure to 10 and 20 min remain constant until reach to concentration 31.25 \u0026micro;g/ml, the viability (%) is 87.29\u0026thinsp;\u0026plusmn;\u0026thinsp;1.02 and 92.19\u0026thinsp;\u0026plusmn;\u0026thinsp;1.95 decrease to 60.13\u0026thinsp;\u0026plusmn;\u0026thinsp;1.11 and 71.31\u0026thinsp;\u0026plusmn;\u0026thinsp;2.14 at concentration 1000 \u0026micro;g/m respectively, as seen in Fig. \u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003e (b). While on HuH-7cells, the cell viability of TiO\u003csub\u003e2\u003c/sub\u003e NPs before and after IR-irradiation remain constant until reach to concentration 62.5 \u0026micro;g/ml. while in case of exposure to 10 and 20 min remain constant until reach to concentration 31.25 \u0026micro;g/ml, the viability (%) is 86.19\u0026thinsp;\u0026plusmn;\u0026thinsp;0.92 and 91.19\u0026thinsp;\u0026plusmn;\u0026thinsp;1.02 decrease to 62.11\u0026thinsp;\u0026plusmn;\u0026thinsp;3.11 and 74.33\u0026thinsp;\u0026plusmn;\u0026thinsp;2.16 at concentration 1000 \u0026micro;g/m respectively. as seen in Fig. \u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003e (b). The values of IC\u003csub\u003e50\u003c/sub\u003e in the case of HuH-7cells and WI38 cells exposed to IR irradiation increased with increasing exposure time. IC\u003csub\u003e50\u003c/sub\u003e of HuH-7cells is 12.8\u0026thinsp;\u0026plusmn;\u0026thinsp;3.2, 60.43\u0026thinsp;\u0026plusmn;\u0026thinsp;7.9 and 80.10\u0026thinsp;\u0026plusmn;\u0026thinsp;21.30 \u0026micro;g/mL for the 5-FU Standard, the control and TIO\u003csub\u003e2\u003c/sub\u003e.NPs after 20 min IR-irradiation respectively, as shown on Fig. \u003cspan class=\"InternalRef\"\u003e7\u003c/span\u003e (b) and supplementary Table 2. While IC\u003csub\u003e50\u003c/sub\u003e of normal WI38 cells showed 65.10\u0026thinsp;\u0026plusmn;\u0026thinsp;9.18, 64.70\u0026thinsp;\u0026plusmn;\u0026thinsp;12.84 and 98.9o\u0026thinsp;\u0026plusmn;\u0026thinsp;20.2o \u0026micro;g/mL for the 5-FU Standard, the control and TIO\u003csub\u003e2\u003c/sub\u003e.NPs after 20 min IR-irradiation respectively. This means that IR -irradiation makes heat to the particles and effect on the poly phenolic acid and make aggregation to the molecules so caused injured to the cell. It should be noted that the statistical variations of minimum, maximum, mean, median, range and standard deviation values of titanium dioxide nanoparticle TiO\u003csub\u003e2\u003c/sub\u003e (NPs) effect on cell viability of HuH-7 cell line and WI-38 cell line before and after exposure to UVC and IR radiation, respectively.\u003c/p\u003e\n \u003cp\u003eIt is necessary to understand some of the limitations and advantages of this approach in the cancer cell line treatment as it needs more study and research to reach better results before clinic stage. Cell lines from either people or non-human animals are used in vitro investigations, where they are combined with the novel drug being developed in a Petri dish or test tube. In vitro research has various advantages. Firstly, the most evident advantage is that they do not have the negative effects of animal testing because they do not damage the person or animal from which the cell cultures were created. Secondly, in vitro approaches have the advantages of being relatively inexpensive to set up and maintain, as well as being dependable, effective, and producing solid results. In contrast to in vitro investigations, in vivo studies are carried out inside a living creature. This occurs in animal test subjects during preclinical studies. \u003cem\u003eIn vivo\u003c/em\u003e experiments can involve either human or animal subjects in clinical trials, the main drawback of in vitro research can be overcome by \u003cem\u003ein vivo\u003c/em\u003e investigations since they can show how a drug affects the body as whole rather than just isolated cells. This makes it possible for in vivo studies to more clearly see potential interactions, which can help them forecast a drug\u0026apos;s safety, toxicity, and effectiveness. This aids scientists in predicting how potential medications will affect human disease. In vivo investigations have their own significant drawbacks despite the fact that they address the problem with in vitro studies. Significant ethical issues surround in vivo research, particularly for preclinical investigations where only animal models are allowed. In future, it is hopeful that we can apply this approach to inject these TIO\u003csub\u003e2\u003c/sub\u003e (NPs) synthesized in a typical procedure of green tea as one of the most antioxidant planta natural products in laboratory mice or larger animals to be activated directly by each of the photons of UVC or IR radiation which confirms the validity of the approach used.\u003c/p\u003e\n \u003cp\u003eTiO\u003csub\u003e2\u003c/sub\u003e nanoparticles were selected from all the data because of their photocatalytic properties, which include: Nanoparticles are well-known Photocatalysts in terms of their mechanism of action. This indicates that they have the ability to absorb light energy, particularly in the ultraviolet spectrum, and use it to produce extremely reactive oxygen species (ROS), like hydroxyl radicals (\u0026bull;OH). Therapeutic uses, their application in photodynamic therapy (PDT) are based on this characteristic. The researchers can cause localized oxidative stress by activating the nanoparticles with a light source (in this case, UVC), which causes lipid peroxidation, protein oxidation, and DNA damage inside the cancer cells. This is a focused strategy to cause the cancerous cells to undergo apoptosis, or programmed cell death. TiO\u003csub\u003e2\u003c/sub\u003e in combination with UVC and nanoparticles are anticipated to create a strong and quick cytotoxic [\u003cspan class=\"CitationRef\"\u003e39\u003c/span\u003e]. The ideal band gap (Eg) of our manufactured nanomaterial, which showed the maximum efficacy, is the \u0026quot;minimum value\u0026quot; that we refer to in our study. This value has physical significance since it controls how well the material absorbs light energy. The material may absorb a wider range of light, including visible light, when its band gap is less (within an ideal range). As a result, when exposed to photo irradiation, more electron-hole pairs (e\u0026minus;/h+) are produced. The formation of Reactive Oxygen Species (ROS) such hydroxyl radicals (\u0026sdot;OH) and superoxide anions (\u0026sdot;O₂⁻) is directly boosted by the higher concentration of charge carriers (electrons and holes) brought about by the smaller band gap. In photocatalysis, these species are principally in charge of breaking down organic contaminants due to their strong oxidizing potential. Thus, the increased photocatalytic activity seen in our studies can be explained by the direct correlation between the lowest band gap value we measured and the highest rate of ROS formation [\u003cspan class=\"CitationRef\"\u003e40\u003c/span\u003e]. The increased cytotoxic effects are explained by the same mechanism. When used in biomedical settings, the effectively produced ROS target essential cellular components in addition to contaminants. These organisms cause extreme oxidative stress in cells, which damages proteins, DNA, and the cell membrane and ultimately results in programmed cell death (apoptosis). This indicates that the strong cytotoxic effects we saw are directly related to the high ROS production efficiency made possible by the ideal minimum band gap [\u003cspan class=\"CitationRef\"\u003e41\u003c/span\u003e]. One important element influencing our TiO\u003csub\u003e2\u003c/sub\u003e nanoparticles\u0026apos; reliable and consistent cytotoxic effects is their size stability. A trustworthy dose-response relationship depends on the surface area-to-volume ratio and photocatalytic activity remaining consistent during the experiment, which is ensured by this stability. The reactive surface area of nanoparticles may decrease as they aggregate or undergo size changes, which might lower the production of Reactive Oxygen Species (ROS) and, consequently, their cytotoxic effectiveness. Consequently, the persistent cytotoxic effects seen in our MTT assay are strongly supported by the stability of our nanoparticles\u0026apos; size [\u003cspan class=\"CitationRef\"\u003e42\u003c/span\u003e]. According to certain research, the in vitro toxicity of nanoparticles can be significantly reduced as they aggregate over time. The significance of regulating physicochemical parameters for reliable and efficient therapeutic applications is thus supported by our findings, which show a steady particle size [\u003cspan class=\"CitationRef\"\u003e43\u003c/span\u003e]. This thorough review article offers a great summary of how agglomeration affects TiO\u003csub\u003e2\u003c/sub\u003e nanoparticle toxicity. It is a great resource to utilize when comparing your results to those of other studies because it addresses findings from a variety of studies. The research highlights that one of the main causes of the diversity in published nanotoxicity data is variations in size and aggregation state.\u003c/p\u003e\n \u003cp\u003eAlthough our results imply that ROS may be the mediator of the observed cytotoxicity, it is crucial to remember that this preliminary investigation did not include direct measurement of ROS levels (for example, using the DCFDA assay). To conclusively validate and measure the role of ROS generation in the mechanistic pathway, more research will be necessary. Our synthetic compound\u0026apos;s noteworthy [e.g., cytotoxic, antibacterial, and anti-inflammatory] properties make it a viable option for a number of potential futures uses: As a Lead Compound for Therapeutics: This study provides evidence of the molecular scaffold\u0026apos;s medicinal potential. The development of a new class of [e.g., anti-bacterial, anti-cancer] drugs may result from future research concentrating on medicinal physics and chemistry efforts to optimize the compound\u0026apos;s structure to increase potency, improve its pharmacokinetic profile, and decrease off-target toxicity. As a tool for basic research: The compound can be used as a chemical probe to study the specific biological pathways it modulates. Understanding its mechanism of action could uncover new therapeutic targets or reveal novel aspects of disease pathology. In combination therapy: Future investigations could explore the synergistic effects of our compound when combined with existing standard drugs. This could potentially lead to more effective treatment regimens that reduce drug resistance and lower required dosages.\u0026quot; As a Basic Research Tool: To investigate the particular biological pathways that the substance affects, it can be employed as a chemical probe. Knowing how it works may help identify new therapeutic targets or new facets of the pathophysiology of the disease. Future research could examine our compound\u0026apos;s synergistic benefits in combination therapy with currently available conventional medications. This may result in more efficient treatment plans that cut dosage requirements and lessen drug resistance.\u003c/p\u003e\n\u003c/div\u003e"},{"header":"5. Conclusion","content":"\u003cp\u003eWe have introduced a new approach of TIO\u003csub\u003e2\u003c/sub\u003e nanoparticles treated by UVC and IR radiation, in vitro, on the growth of HuH-7 cells (human Hepatocellular cancer cell line), to obtain a unique technique for anticancer activity over exposure periods of 0.5\u0026ndash;10 h of UVC radiation and from 2.5\u0026ndash;20 min of IR radiation, respectively. The obtained results suggest that the extent of UVC-induced TIO\u003csub\u003e2\u003c/sub\u003e nanoparticles\u0026rsquo; activation is dose-dependent, whereas in the case of IR treatment it shows non similar trend. Our results imply that this new technique enhances TIO\u003csub\u003e2\u003c/sub\u003e nanoparticles efficiency treated by UVC radiation, allowing high-degree treatment of cancer cells. UVC treatment has proven to be the most efficient process in the current study, which induces continuous steady decreases in the values of IC\u003csub\u003e50\u003c/sub\u003e of HuH-7 cells, and a highest increase in the values of WI-38 cell line. The decrease in the optical band-gap energy as a result of UVC exposure at 4 h interval period can be mainly ascribed to the formation of unsaturated bonds, in which π-π * transitions of delocalized electrons can occur. The high absorption of UVC light by the molecules and atoms of malignant cells, particularly in the far-UVC region, is attributed to absorption by peptide bonds of proteins and lipids, and which form an inverted resonance peak.\u003c/p\u003e \u003cp\u003eTo our knowledge, the cytotoxic effects of TIO\u003csub\u003e2\u003c/sub\u003e nanoparticles treated by UVC, in vitro, on the growth of HuH-7 cells (human Hepatocellular cancer cell line has never been reported before. The optical band gap energy measurements clearly confirm that the positive changes brought to the malignant cells as a result of UVC exposure are physico-biological in nature, especially when minimize the values of these optical band gap energies.\u003c/p\u003e \u003cp\u003eFinally, we concluded that the chemotherapy gives relatively better result than our used method, but it has its drawbacks, which is that after a period of time, it can cause death, so we resorted to UVC radiation therapy, which may cause less harm to normal cells. This finding agrees well with the published data in the literature on both chemotherapy and radiotherapy which also support the powerful accuracy of the method used.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no competing interests.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthical approval\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent to participate\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent to publish\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eA.F. Saad: Conceptualization, Methodology, Writing - Reviewing, Editing; Yasser A. Selim: Conceptualization, Methodology, Writing - Reviewing; Mona Hamada: Data curation, Investigation, Formal analysis, Writing-original draft; Mohammed Elywa: Co-supervision, Investigation\u003c/p\u003e\u003ch2\u003eData Availability\u003c/h2\u003e\u003cp\u003eAll data generated or analyzed during this study are included in this published article. The datasets generated during and/or analyzed during the current study are available from the corresponding author on reasonable request.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eYu M, Zhiying S, Johnny V, Orit K, Andrew Z (2016) W Cancer Nanatechnol. ; 7\u0026ndash;11\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHogle WP (2006) The state of the art in radiation therapy. Semin Oncol Nurs 22:212\u0026ndash;220\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMallick I, Waldron JN (2009) Radiation therapy for head and neck cancers. 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Adv Sci 11(3):2304918\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"discover-nano","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"narl","sideBox":"Learn more about [Discover Nano](https://www.springer.com/journal/11671)","snPcode":"11671","submissionUrl":"https://submission.nature.com/new-submission/11671/3","title":"Discover Nano","twitterHandle":"@SpringerOpen","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Discover Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"TIO2 nanoparticles, Photocatalysts. UVC-irradiation, IR- irradiation, cytotoxic effects, Cancer cell line treatment, anticancer activity","lastPublishedDoi":"10.21203/rs.3.rs-8373569/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8373569/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eThe aim of this study is to describe the effect of TiO₂ nanoparticles (NPs) synthesized with green tea extract, as a natural product on cancer cell lines, for example, the human hepatocellular cancer cell line, and their interaction with phototherapies such as ultraviolet (UV) and infrared (IR) therapy for anticancer treatment. TiO₂ (NPs) was synthesized in a procedure of green tea as one of the most antioxidant plant-derived natural products. Titanium dioxide nanoparticles (TiO₂ NPs) samples were exposed to UVC and IR radiation. The qualitative impacts of these treatments have been analyzed by means of ultraviolet/visible (UV/Vis) spectrophotometry in the range 200\u0026ndash;800 nm, considering optical properties, as well as the cytotoxic effect, which was evaluated by measuring the numbers of viable cells, which were determined by the MTT test. The use of TiO₂ in conjunction with ultraviolet or infrared radiation has shown a remarkable ability to enhance the killing of cancer cells by generating reactive oxygen species. Therefore, the TiO₂ group and the activating treatment showed great potential and could be an effective therapeutic strategy against cancer. The final treatment results proved to be highly dependent on the specific order of the UV dose and had a lesser effect in the case of IR radiation. The results clearly showed that the value for the IC50 of cell viability of the HuH-7 cell line, which equals 36.40\u0026thinsp;\u0026plusmn;\u0026thinsp;7.80 \u0026micro;g/ml, at 4 h UVC exposure was significantly lower than the IC\u003csub\u003e50\u003c/sub\u003e values for the other UVC exposure interval times. In addition, the exposure at the same dose had a better effect on normal cells (WI-38 cell line) and provided a greater value for this IC\u003csub\u003e50\u003c/sub\u003e parameter compared to both samples of control and standardized chemotherapy, whereas the IR treatment did not have a similar trend. The results also demonstrate that the production of TiO₂ nanoparticles by green synthesis with biological extracts is an effective technique to produce non-toxic materials for cancer treatment. Despite the good results achieved from using nanotechnology in medicinal treatment, it is essential that clinical applications remain limited, requiring additional studies on live animals that resemble the human body.\u003c/p\u003e","manuscriptTitle":"Titanium dioxide nanoparticles treated by UVC and IR radiation-induced positive changes in a cancer cell line treatment","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-01-16 08:22:12","doi":"10.21203/rs.3.rs-8373569/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2026-05-11T19:33:27+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-03-05T21:03:30+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-03-02T10:50:07+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"178988786455024468505160802826480371929","date":"2026-02-26T07:57:55+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"143556183319988401364521787840578494168","date":"2026-02-24T09:19:05+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"138395396399369646958665206437482464092","date":"2026-02-23T14:02:46+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"317943070444326819276249334838628693180","date":"2026-02-23T12:07:32+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"131014821763542287087196108957906440954","date":"2026-02-23T11:42:06+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-01-14T11:00:49+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"317737400190775805159971663176231832753","date":"2026-01-14T10:36:21+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2026-01-09T09:30:29+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-12-29T05:51:28+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-12-28T13:45:39+00:00","index":"","fulltext":""},{"type":"submitted","content":"Discover Nano","date":"2025-12-28T13:37:25+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"discover-nano","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"narl","sideBox":"Learn more about [Discover Nano](https://www.springer.com/journal/11671)","snPcode":"11671","submissionUrl":"https://submission.nature.com/new-submission/11671/3","title":"Discover Nano","twitterHandle":"@SpringerOpen","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Discover Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"b4e2b648-0d7c-419c-997b-760d3d25acef","owner":[],"postedDate":"January 16th, 2026","published":true,"recentEditorialEvents":[{"type":"decision","content":"Revision requested","date":"2026-05-11T19:33:27+00:00","index":"","fulltext":""}],"rejectedJournal":[],"revision":"","amendment":"","status":"in-revision","subjectAreas":[],"tags":[],"updatedAt":"2026-05-11T19:40:00+00:00","versionOfRecord":[],"versionCreatedAt":"2026-01-16 08:22:12","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8373569","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8373569","identity":"rs-8373569","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}