The potential of HSA-stabilized zinc oxide nanoparticles as radiosensitizers to enhance the cytotoxic effects and radiosensitivity of cervical cancer cells

Abstract

Background

Cervical cancer is a significant cause of death among women worldwide, and limited treatment approaches are available for patients with metastatic or recurrent disease. Recently, the combination of nanoparticles (NPs) and radiotherapy (RT) has been shown to be an effective treatment because it enhances the sensitivity of cancer cells to radiation. ZnO NPs stabilized with HSA have become one of the most popular types of metal oxide nanoparticles because of their cost-effectiveness and minimal toxicity. Therefore, our study aimed to investigate the radiosensitization effects of HSA/ZnO-NPs on cervical (HeLa) cancer cells under megavoltage (MV) X-ray irradiation.

Methods

HSA/ZnO-NPs were prepared and characterized by SEM and Dynamic Light Scattering (DLS) technique. The cytotoxicity of HSA/ZnO NPs was evaluated in HeLa cells via an MTT assay. The radiosensitization effects were investigated under megavoltage X-ray irradiation using a clonogenic survival assay and quantifying γH2AX foci. Moreover, apoptosis and cell cycle analyses were conducted using a Muse^™ Cell Analyzer.

Results

HSA/ZnO-NPs reduced the viability of HeLa cells in a dose-dependent manner, which revealed that the IC50 of HSA/ZnO-NPs was approximately 30 µg/mL. The prepared particles exhibited moderate aggregation regarding hydrodynamic size (approximately 300–400 nm) and a negative zeta potential charge. Compared to the control group, combining HSA/ZnO-NPs with irradiation reduced the colony-forming ability and survival of HeLa cells by approximately 51% and 71% for 2 and 4 Gy, respectively. Correspondingly, the results of the apoptosis analysis showed that combining HSA/ZnO-NPs with irradiation significantly increased apoptosis induction by approximately 39.15% and 77.67% for 2 and 4 Gy, respectively. In addition, we observed a significant increase in cell cycle arrest at the S phase, by about 11.3% and 19.3% for 2 and 4 Gy, respectively.

Conclusions

HSA/ZnO-NPs could significantly enhance the cytotoxic effects of ionizing radiation, which suggests the promising potential of cervical cancer radiotherapy under megavoltage X-ray irradiation.

Introduction

Cervical cancer is the most common cause of cancer-related death in women. From 2012 to 2018, a notable increase in both the number of deaths and the incidence of newly diagnosed cervical cancer cases was observed worldwide. (Bray et al. 2018; Arbyn et al. 2020) Radiotherapy (RT) is a crucial curative or adjuvant treatment approach that involves the precise targeting of tumor tissue with high doses of ionizing radiation, which destroys cancer cells. (Landoni et al. 2017) However, the disadvantages and limitations of RT include the possibility of damaging the surrounding normal tissue and the possibility that some tumor cells are located farther from the radiation site and thus receive a lower radiation beam intensity. Moreover, radioresistance of tumors is a significant contributing factor to poor prognosis in cancer patients and the primary reason for radiotherapy failure, which can eventually cause tumor recurrence and metastasis. (Liu et al. 2021b).

A promising approach to enhance the tumor response and reduce average tissue damage is the combination of RT and radiosensitizers, which are therapeutic or otherwise inert agents that increase the radiosensitivity of tumor cells. For example, cisplatin and its Pt analogs are the most frequently used drugs in concomitant chemoradiation therapy. Although cisplatin is used as a radiosensitizer for locally advanced cervical cancer, patients often develop increased toxicity-related problems, such as hematologic and gastrointestinal complications. However, the clinical efficacy of existing radiosensitizers for the treatment of cervical cancer is not completely understood. (Candelaria et al. 2006) Therefore, discovering potential novel, less toxic, and more effective radiosensitizers against cancer is crucial to improving treatment efficacy.

Advanced nanotechnology and nanomedicine have emerged over the past decade and have created valuable opportunities for enhancing tumor radiosensitization. (Liu et al. 2021a; Song et al. 2023) As a result, researchers are exploring methods to improve radiotherapy-based cancer treatments using nanomaterials to increase the sensitivity of cancer cells to radiation and to overcome the challenge of tumor radioresistance. To develop novel and effective cancer treatment regimens, scientists are utilizing the unique properties of nanomaterials, including the following: enhanced cellular uptake, ease of functionalization, unique optical, electronic, and magnetic properties, distinct biodistribution and pharmacokinetics, enhanced permeability and retention (EPR) effects, controlled release mechanisms, large surface areas, stability, and customizable features, such as size, shape, and surface charge.(Zhang et al. 2022; Arif et al. 2023) Therefore, new nano-radiosensitizers are continuously being developed and proposed.

Zinc oxide nanoparticles (ZnO NPs) are semiconductor materials with wide biomedical applications, including bioimaging, biosensing, drug delivery, and cancer treatment. ZnO NPs have attracted interest for use in various biomedical applications because of their high stability and inherent photoluminescence, which are helpful for biosensing, and their broad bandgap semiconductor characteristics, which are beneficial in photocatalytic systems. (Mishra et al. 2017) Finally, they have the ability to induce ROS generation, which leads to inherent selective toxicity toward cancer cells. (Jiang et al. 2018) Although ZnO NPs are semiconductor materials that are environmentally friendly, their nanoscale size can enable selective tumor cell destruction due to their cytotoxic effects. (Rasmussen et al. 2010) Therefore, these inorganic nanoparticles require proper functionalization for biomedical applications, especially those related to bioimaging and drug delivery. A previous study reported that surface-functionalized ZnO NPs with BSA functionalized with 3-CCA have highly biocompatible properties, which indicates that they are more useful for various biological and industrial applications. (Kanchana and Mathew 2021).

Furthermore, the functionalization or coating of nanoparticles with targeting agents, such as proteins, DNA, and antibodies, may further increase their suitability for biomedical applications. One of the proteins extensively used for drug delivery is human serum albumin (HSA). HSA is a protein predominantly found in plasma (60% of the total protein in plasma), is present in human blood at a concentration of 35–50 g/L and has a long half-life of 19 days. It contains 585 amino acids and has a molecular weight of 66.4 kDa. (Al-Harthi et al. 2019) HSA can transport fatty acids, bile pigments, amino acids, steroid hormones, and metal ions within the body. These diverse physiological roles highlight its potential as a drug carrier. Recently, significant interest was generated in using HSA as a drug carrier because of its excellent biocompatibility, stability, high capacity for drug delivery, targeting ability, long half-life, and easy accessibility. (Bhushan et al. 2017; Tao et al. 2021).

Therefore, in this study, we examined the potential of ZnO-NPs stabilized with HSA (HSA/ZnO-NPs) for use as radiosensitizers to improve the efficacy of radiation therapy. Cervical cancer cells (HeLa cells) were subjected to ionizing radiation in the presence or absence of HAS/ZnO-NPs. We then investigated the cytotoxic and radiosensitizing effects of HSA/ZnO-NPs on cell viability, colony formation, the cell cycle, and apoptosis.

Materials and methods

Preparation of HSA/ZnO-NPs

Zinc oxide nanopowder (< 100 nm particle size; CAS Number: 1314-13-2) was obtained from Sigma‒Aldrich (St. Louis, Missouri, MI, USA). The particle mixture was prepared as follows to obtain good particle dispersion. Briefly, 2 mg of ZnO-NPs was dissolved in 850 mL of distilled water and then sonicated with an ultrasonic homogenizer (Omni International Sonic Ruptor 250, Kennesaw, GA, USA) at 30% power for 60 s in continuous mode. Then, 50 µL of 10% human serum albumin (HSA; Sigma‒Aldrich. St. Louis, MO, USA) was added to stabilize the suspension. The salt concentrations were reached by adding 100 µL of 10X phosphate-buffered saline (PBS, Thermo Fisher Scientific, Vilnius, Lithuania) with a physiological pH of 7.4. The stock suspension was further diluted to achieve various concentrations of HSA/ZnO-NPs for the experiments.

Characterization of HSA/ZnO-NPs

Particle size and zeta potential

The hydrodynamic size of nanoparticles was measured using Dynamic Light Scattering (DLS) with a Zeta-Sizer Ultra (Malvern Instruments Ltd., Malvern, UK). All measurements were performed at a controlled temperature of 25 °C. Before each measurement, samples were equilibrated for 120 s to ensure stability. Each sample was analyzed in triplicate, allowing for calculating the average size and size distribution, represented by the polydispersity index (PDI), using the instrument's software. For zeta potential measurements, the nanoparticle suspension was diluted with distilled water to achieve the appropriate concentration, ensuring a conductivity range of 0.01–0.04 mS/cm. These measurements were also conducted at 25 °C, with the pH adjusted to 7.0. To ensure the accuracy of the results, each sample underwent a minimum of three repeat measurements. The Smoluchowski model was applied for data analysis, relating the measured electrophoretic mobility to zeta potential.

Scanning electron microscopy (SEM)

The morphology of the HSA/ZnO-NPs was analyzed using a JSM-5910 SEM (JEOL USA; Peabody, MA, USA). The samples were diluted in distilled water, and a drop of the suspension was placed on copper tape, which was allowed to dry at room temperature overnight. After drying, the samples were coated with a thin layer of gold. SEM images were obtained under a high vacuum at an operating voltage of 15 kV.

Cell culture

The HeLa cell line was a kind gift from Assistant Professor Dr. Nipaporn Sankuntaw from Thammasat University, Thailand. Cells were cultured in Dulbecco's Modified Eagle's Medium (DMEM, Life Technologies Corporation, NY, USA) supplemented with 10% fetal bovine serum (FBS, Life Technologies Corporation, NY, USA) and 1% penicillin/streptomycin (HyClone Laboratories Inc., Logan, Utah) and cultured at 37 °C in a humidified incubator with 95% air and 5% CO₂.

Cell viability

The viability of HeLa cells treated with HSA/ZnO-NPs in vitro was assessed via a 3-(4,5-dimethylthiazol-2-yl)−2,5-diphenyltetrazolium bromide (MTT, Sigma Chemical Co., St. Louis, MO, USA) assay. Briefly, 1 × 10⁴ HeLa cells were seeded in a 96-well plate with 100 μL of DMEM per well and incubated overnight at 37 °C with 5% CO2 to allow the cells to reach confluence. Subsequently, the medium was replaced with 100 μL of fresh medium containing either PBS as a control or HSA/ZnO-NPs at an IC50 concentration of 30 μg/mL. After incubation for 24 h, 100 μL of 5 mg/mL MTT in serum-free DMEM was added to each well. After three additional hours of incubation, the medium was replaced with 100 μL of DMSO. The plate was then gently shaken to dissolve the insoluble formazan crystals, and the absorbance of each well was determined via a SpectraMax i3x microplate reader (Molecular Devices, Sunnyvale, CA, USA) at 570 nm. The mean and standard deviation for three parallel wells for each sample were reported.

Irradiation

Photon beam irradiation was performed via a 6 MV linear accelerator (Varian Clinac 2100C, Varian Medical System, Palo Alto, CA, USA) from the Faculty of Medicine, Naresuan University, with single doses of 2 and 4 Gray (Gy) at room temperature. The source-to-sample distance was 100 cm. The 6-cm culture dishes containing HeLa cells were irradiated with a vertical beam in a 20 × 20 cm² field. The dishes were placed horizontally at the isocenter within a bolus phantom at a depth of 3 cm.

Colony formation assay

The effects of HSA/ZnO-NPs on the survival of HeLa cells after treatment and irradiation were investigated using a clonogenic survival assay. After 2 × 10⁵ HeLa cells were seeded in a 6-cm cell culture dish, they were treated with HSA/ZnO-NPs for 24 h. Next, X-ray irradiation was administered at 2 and 4 Gy. Immediately following treatment, the cells were harvested via trypsinization, washed with PBS, and then counted. Irradiated cells were then plated homogeneously in a 6-well plate at a density of 200 cells per well and incubated in a humidified atmosphere (5% CO₂, 37 °C) for 10–14 days. For HSA/ZnO-NP treatment combined with irradiation, the cells were plated at a density of 400 cells per well. The cells were fixed in 4% paraformaldehyde and stained with crystal violet. Colonies with at least 50 cells were counted. The number of colonies that grew and the relative survival of the colonies were expressed as the surviving fraction (SF) according to the following formula (Franken et al. 2006):

$$\text{SF} = \frac{\text{No. of colonies formed after treatment}}{\text{No. of cells seeded }{\text{X}}\text{ PE}}$$

in which:

$$\text{PE }\left(\text{plating efficiency}\right)\text{=}\frac{\text{No. of colonies formed}}{\text{No. of cells seeded}}\text{ X 100\%}$$

Moreover, the sensitivity enhancement ratio (SER) plays a crucial role in evaluating the effectiveness of a radiosensitizing agent. The SER of HSA/ZnO NPs was calculated according to the following equation (Mohammadian et al. 2022):

$$\text{SER} = \frac{\text{D50 (without sensitizer)}}{\text{D50 (with se}\text{nsitizer)}}$$

Immunofluorescence staining

The detection of γ-H2AX foci was performed using immunofluorescence staining. HeLa cells were cultured on coverslips in 6-well plates and pre-incubated with either 30 µg/mL HSA/ZnO nanoparticles or with DMEM as a control for 24 h before irradiation. Following irradiation, the cells were harvested and rinsed with PBS. Cells were fixed in 4% paraformaldehyde for 15 min, permeabilized with 0.1% Triton X-100, and blocked in PBS containing 2% bovine serum albumin (BSA). To detect γ-H2AX, cells were incubated overnight at 4 °C with a phosphohistone-H2AX (Ser139) monoclonal antibody (diluted 1:800, Thermo Fisher Scientific, Rockford, IL, USA). After rinsing in blocking buffer, cells were stained in the dark with an Alexa-Fluor-488 conjugated polyclonal goat anti-mouse IgG antibody (diluted 1:1000, Life Technologies, Eugene, OR, USA) for 1 h. The slides were washed with PBS and mounted with a medium containing DAPI (4', 6-diamidino-2-phenylindole) for nuclear staining (Life Technologies, Eugene, OR, USA). Fluorescence images were captured using a Carl Zeiss fluorescence microscope (Carl Zeiss AG, Oberkochen, Germany).

Cell cycle analysis

HeLa cells were cultured in a 6-cm cell culture dish for 24 h and incubated with 30 µg/mL HSA/ZnO-NPs for 24 h before irradiation with 2 and 4 Gy radiation doses. The cells treated with only the culture medium served as control. The cells were collected, transferred to a 1.5 mL microcentrifuge tube, and washed twice with PBS. They were then fixed in 1 mL of cold 70% ethanol for 5 h. A total of 200 µL of each cell suspension (10⁶ cells/mL) was washed with 250 µL of PBS and then with 200 µL of Muse^™ cell cycle reagent and then incubated in the dark for 20 min at room temperature, after which the cells were analyzed via a Muse™ Cell Analyzer (Merck, Darmstadt, Germany).

Apoptosis analysis

HeLa cells were cultured in a 6-cm cell culture dish for 24 h and incubated with 30 µg/mL HSA/ZnO-NPs for 24 h before irradiation with 2 and 4 Gy radiation doses. The cells treated with only culture medium served as the control. After harvesting, the cells were transferred to a 1.5 mL microcentrifuge tube and washed twice with PBS. Next, 100 µL of Muse^™ Annexin V and Dead Cell Reagent was mixed with 100 µL of the cell suspension (5 × 10⁵ cells/mL) and incubated in the dark for 20 min at room temperature. Apoptotic cells were then analyzed using a Muse^™ Cell Analyzer (Merck, Darmstadt, Germany) and gated on Annexin V-FITC-positive cells and 7-AAD-positive cells.

Statistical analysis

All the experiments were performed in triplicate, and the data are expressed as the means ± SEMs. For all statistical analyses, one-way analysis of variance (ANOVA) was performed using GraphPad Prism software (version 10). A p value < 0.05 indicates statistical significance.

Results

Characterization of HSA/ZnO-NPs

The mean diameter of the HSA/ZnO-NPs aggregated and assessed in distilled water was approximately 300–400 nm, with a PDI of 0.337, and the zeta potential was −19 ± 0.74 mV. The particles showed a morphology of agglomerates and aggregates, but the size of the primary particles must be smaller than 100 nm, as shown in Fig. 1. Once the particles were stabilized with HSA, the HSA/ZnO-NPs produced more stable colloidal suspensions with negative zeta potential charges than ZnO alone.

Fig. 1

A SEM image displaying the size and morphology of the ZnO-NPs and photographic of particle suspension in PBS within 5 min after preparation. B Particle size and zeta potential of the HSA/ZnO NPs. C Cytotoxic effects of HSA/ZnO NPs on HeLa cells. Significant differences are indicated by *p < 0.0001 compared with the control group

Cytotoxicity of HSA/ZnO-NPs in HeLa cells

The percentages of viable (% cell viability) HeLa cells treated with different concentrations of HSA/ZnO-NPs gradually decreased. In contrast, the toxicity percentages increased in a dose-dependent, as illustrated in Fig. 1. After incubation with HSA/ZnO-NPs for 24 h, a significant decrease in cell viability was determined at concentrations of 30 µg/mL and higher. The median growth inhibitory concentration (IC50) of HSA/ZnO-NPs was approximately 30 µg/mL in HeLa cells.

Colony formation assay of HeLa cells treated with HSA/ZnO-NPs under irradiation

After 2 and 4 Gy irradiation, clonogenic survival was notably reduced in all the groups compared with the nonirradiated HeLa cells (control group), as demonstrated in Fig. 2. Compared with the control, the combination of irradiation and HSA/ZnO-NPs at a concentration of 30 µg/mL significantly reduced cell survival by 51% for the 2 Gy with HSA/ZnO-NP group and 71% for the 4 Gy irradiation with HSA/ZnO-NP group. The SER of HSA/ZnO-NPs was approximately 1.3 (Koch et al. 2010). These findings indicate that nanoparticles can increase the damage caused by ionizing radiation. These results reveal that HSA/ZnO-NPs have significant radiosensitization potential in HeLa cells.

Fig. 2

A Clonogenic assay and % plating efficiency of HeLa cells treated with 30 µg/mL HSA/ZnO-NPs for 24 h before irradiation (IR) (2, 4 Gy); the number of clones was counted 14 days after IR. B Clonogenic survival analysis also demonstrated a reduction in survival. All the experiments were repeated three times. The error bars represent the standard error of the mean

γH2AX analysis using fluorescent microscopy

Detecting γ-H2AX foci via immunofluorescence staining is crucial for assessing DNA double-strand breaks (DSBs). (Valente et al. 2022; Takano et al. 2023) The accumulation of γ-H2AX at the sites of DSBs, forming nuclear foci, indicates DNA damage. We assessed γ-H2AX foci by immunofluorescence in HeLa cells following exposure to 2 and 4 Gy irradiation and combination with HSA/ZnO-NPs, as shown in Fig. 3. The control group exhibited the lowest count of positive green cells, while the HSA/ZnO-NPs exposure and irradiation-alone groups showed notable presence of positive green cells. However, the number of positive green cells increased in the HSA/ZnO-NP combined with 2 or 4 Gy radiation groups compared to each irradiation-alone group in a radiation-dependent manner. These findings indicate that HSA/ZnO-NP could enhance the radiation-induced DSBs by leading to a delay in the DNA repair process.

Fig. 3

Detection of γ-H2AX foci via immunofluorescence staining. In untreated cells (control), γ-H2AX foci were most undetected, followed by a notable increase only in irradiation. In contrast, combining treatment between HSA/ZnO-NPs and irradiation exhibited persistent γ-H2AX foci, which were almost prominent and delectable in a radiation-dependent manner. These observations are illustrated for HeLa cell lines, highlighting the potential role of combining treatment between HSA/ZnO-NPs and irradiation in extending DNA damage markers and possibly enhancing cellular radiosensitivity

Influence of HSA/ZnO-NPs concentration and irradiation on the cell cycle distribution of HeLa cells

The effect of HSA/ZnO-NP treatment on the cell cycle distribution of HeLa cells was examined via the Muse Cell Analyzer and the Muse^® Cell Cycle Assay Kit. We determined the cell cycle distribution in HeLa cells to gain further insight into the radiosensitization mechanism of HSA/ZnO-NPs. As shown in Fig. 4, the percentage of cells in G0/G1 phase decreased, whereas that of cells in S phase increased in all the treatment groups compared with the control group. The percentage of cells in S phase in the 30 µg/mL HSA/ZnO-NP treatment group was slightly more significant than that in the control group but was not different from that in the irradiation treatment groups. However, the percentage of cells arrested in S phase was dramatically increased in a radiation dose-dependent manner in the combined treatment group compared with the control group (p < 0.05), which received HSA/ZnO-NPs or irradiation alone.

Fig. 4

Cell cycle analysis of HeLa cells was conducted using the Muse™ Cell Analyzer after various treatments; the histogram plots illustrate the percentage of cells distributed across different phases. The data are presented as the means ± SEMs of three independent experiments. Significant differences are indicated by *P < 0.05, ***P < 0.001, ****P < 0.0001 compared with the control group

Characterization of cell death induced by HSA/ZnO-NPs and irradiation

To determine the percentage of HeLa cells that died after irradiation combined with HSA/ZnO NPs, the cells were stained with Annexin V & Dead Cell Reagent and analyzed using the appropriate program on the Muse Cell Analyzer. For Annexin V and PI, positive cells are presented as apoptotic and necrotic cells, respectively.

Figure 5 shows that the percentage of apoptotic cells in the 30 µg/mL HSA/ZnO-NP treatment group was significantly more significant than in the control and irradiation treatment groups. Moreover, the percentage of apoptotic cells increased dramatically in a radiation dose-dependent manner in the combined treatment group compared with the control group. Interestingly, HSA/ZnO-NPs induced apoptosis and necrosis in HeLa cells in contrast to irradiation alone, resulting in an enhanced radiation ability to induce apoptosis instead.

Fig. 5

Apoptotic ratio of the cells was detected at 24 h after treatment with 30 µg/mL HSA/ZnO-NPs and radiation. The numbers in the plots indicate the percentage of positive cells for the indicated markers. The apoptotic rate is represented as the percentage of Annexin V-positive cells. The data are presented as the means ± SEMs of three independent experiments. Significant differences are indicated by **P < 0.01, ***P < 0.001, and ****P < 0.0001 compared with the control group

Discussion

Radiotherapy is the most effective and widely recognized approach to cancer treatment. However, the limitation of ionizing radiation poses a significant challenge because it can potentially harm normal surrounding tissues. Recently, various nanoparticles have demonstrated the ability to enhance the effectiveness of radiation treatment by directly interacting with ionizing radiation to increase the sensitivity of cells to radiation. (Arif et al. 2023) This study examined whether HSA/ZnO-NPs could be potential radiosensitizers to enhance the radiation treatment effect in HeLa cells.

The sizes of the HSA/ZnO-NPs were determined by SEM and were confirmed by dynamic light scattering (DLS). The HSA/ZnO-NPs were aggregated particles with an average hydrodynamic diameter of approximately 300–400 nm and a moderate PDI. The resultant particle size was found to be larger than the commercially supplied value (< 100 nm particle size). The difference results from the measurement of dynamic light scattering, which involves assessing the Brownian motion and size distribution of particles within a solution, resulting in a mean hydrodynamic diameter that is usually larger. (Alarifi et al. 2013) As we know, the size-dependent properties of nanoparticles (NPs) are critical in determining their effectiveness as radiosensitizing agents. For instance, The study concludes that small sizes of (12.1 and 27.3 nm) PEG-coated Au NPs exhibit higher toxicity and enhanced radiotherapy effects than larger sizes, demonstrating significant potential for applications in cancer treatment and drug delivery with minimal toxicity. (Garrigós et al. 2024). In contrast, while smaller PEG-coated Au NPs demonstrate enhanced therapeutic effects, larger NPs may still hold value in specific applications where slower release or prolonged circulation time is desired, suggesting a need for tailored approaches in NP-based therapies. However, further studies have investigated the size-dependent relationship between NP behavior and therapeutic outcomes.

Moreover, the surface charge, known as zeta potential, represents a significant characteristic of nanoparticles. The zeta potential indicates how nanoparticles interact with one another and the medium into which they are introduced. As the zeta potential increases in aqueous solutions, the repulsive forces between nanoparticles also increase, which leads to more stable particles with a more uniform size distribution. This stability is crucial for preventing aggregation and subsequent precipitation and ensures a stable colloidal dispersion of the nanoparticles. A zeta potential value of ± 30 mV is commonly regarded as the stability threshold. However, colloids tend to become unstable below this level and are more likely to undergo rapid coagulation. (Hunter 1988; McNeil 2011) Our experiments revealed that the colloidal stability of HSA/ZnO-NPs was greatly improved. Compared with that of the naked ZnO NPs, the zeta potential of the HSA/ZnO-NPs was approximately −23 mV, and this value increased with a more negative change in water. This finding indicates that the presence of HSA could influence the colloidal aspects of the ZnO-NPs. These results support the concept that HSA can prevent the aggregation of ZnO NPs.

Previous studies have reported that ZnO-NPs can be coated and combined with biological molecules (e.g., serum albumin). (Žukiene and Snitka 2015) In practice, the interaction of NPs with biological molecules, such as proteins, reduces the free surface energy and increases the stability of NPs. In this study, we aimed to enhance the dispersibility and minimize the aggregation of ZnO-NPs by stabilizing them with human serum albumin (HSA). As the most abundant protein in the circulatory system, comprising approximately 60% of plasma proteins, HSA plays a key role in binding a variety of biomolecules, including metabolites, drugs, organic compounds, and both metallic and non-metallic nanoparticles.(Kannan et al. 2011; Paul et al. 2022). Further investigation is warranted to rigorously assess the advantages of HSA-modified ZnO-NPs compared to unmodified ZnO. In addition, in-depth characterization of the HSA coating on ZnO-NPs, including analyses through FTIR spectroscopy, is necessary to strengthen our findings and confirm the effectiveness of the stabilization process.

The cytotoxicity of HSA/ZnO-NPs, as determined by an MTT assay, revealed that our particles can induce dose-dependent toxicity. Previous studies have investigated the potential toxicity of ZnO-NPs. Some findings reported by Chakraborti et al. highlight the potential toxicity of ZnO-NPs in different cell lines (Chakraborti et al. 2017) and in animals through different mechanisms. However, our study utilized only a single cell line, and further research incorporating at least two cell lines, including non-cancerous cells, is recommended to provide a more comprehensive understanding of toxicity and therapeutic potential. Moreover, according to Zhu et al., the primary toxic effects are driven by the generation of reactive oxygen species (ROS), which lead to oxidative stress, lipid peroxidation, and damage to cell membranes. (Zhu et al. 2019) Importantly, these toxic effects can arise from ZnO-NPs or dissolved zinc ions. Free zinc is already harmful to humans at high doses. (Agnew and Slesinger 2022) However, the mechanisms involving free zinc ions and ZnO-NPs should be considered and analyzed independently. Therefore, when conducting further research on ZnO-NPs, the incorporation of zinc salt as a control is essential.

In this study, HSA/ZnO-NPs were shown to reduce the survival of HeLa cells after combined treatment with irradiation. The radiosensitivity of HSA/ZnO-NPs was evaluated via a colony formation assay. Compared with irradiation alone, the combination of HSA/ZnO-NPs and irradiation markedly reduced the surviving fraction of HeLa cells, and the impact of this combination on the survival of HeLa cells was greater than that of irradiation alone (Fig. 2). A synergistic effect can be observed when both treatments are applied concurrently; this implies that the combined treatment involving irradiation and HSA/ZnO-NPs was more potent than their individual effects. Consequently, HSA/ZnO-NPs may be a promising adjunct therapy to enhance radiotherapy. The use of HSA/ZnO-NPs could allow for a reduction in the radiation dosage while achieving a comparable level of tumor cell death.

A significant approach to nanoparticle-sensitized radiotherapy involves regulating the cell cycle in cancer cells. The cell cycle consists of interphase (G1, S, and G2 phases) and mitosis (M). During G1 phase, cells grow, produce RNA, and synthesize proteins necessary for DNA replication. Under normal conditions, DNA replication occurs during S phase, while cells continue to grow and synthesize new proteins in G2 phase. M stage involves the division of the nucleus and cytoplasm, completing the cell cycle. (Joiner and van der Kogel 2018) The DNA damage checkpoint follows all cell cycle phases to prevent abnormal genetic materials from passing to the next generation. After DNA damage is detected, the cell will become arrested for repair or to induce cell death if the damage is sufficient. (Hall and Giacci 2018) NP treatment usually has various effects on cancer cell DNA damage, which results in a change in the cell cycle distribution pattern. Many studies have shown that the number of cells in G0/G1 phase decreases, whereas the number of cells in S or G2/M phase increases. (Rosa et al. 2017) These are hallmarks of cells that present with DNA damage. Previous studies have demonstrated that ZnO-NPs have anti-cancer effects on breast cancer MCF-7 cells. The underlying mechanisms include the induction of cell cycle arrest and apoptosis (Moghaddam et al., 2017). According to our results, after 24 h of HSA/ZnO-NP treatment, the number of cells in the G2/M phase tended to increase. However, combining 2 or 4 Gy irradiation with HSA/ZnO-NP significantly decreased the percentage of cells in the G0/G1 phase. In contrast, the number of cells in the S phase significantly increased compared to the control or irradiation alone (Fig. 4). We investigated the radiosensitizing mechanism of HSA/ZnO-NP by evaluating DSB damage and DNA repair by quantifying γ-H2AX foci, a marker of DSB damage. (Redon et al. 2010) The results demonstrated that HSA/ZnO-NP combined with 2 or 4 Gy irradiation increased the formation of irradiation-induced γ-H2AX foci and delayed their degradation compared to treatment with HSA/ZnO-NP or irradiation alone (Fig. 3). These results suggest that HSA/ZnO-NP weakens DNA, enhancing double-strand breaks (DSBs) during irradiation. Our findings align with those of Shan et al., who demonstrated that zinc combined with clioquinol can inhibit the NF-κB-ATM pathway (Lu et al. 2018), which plays a key role in DNA damage response and homologous recombination (HR) repair during the S/G2 phase of the cell cycle, resulting in increased DNA damage and impaired DSB repair (Karl et al. 2009) In addition, cells in the G0/G1 phase, despite being damaged by zinc and radiation, can still repair themselves through the non-homologous end joining (NHEJ) pathway and progress into the S phase. Taken together, the prolonged presence of γ-H2AX foci in cells treated with HSA/ZnO-NP combined with 2 or 4 Gy irradiation is due to the inhibition of ATM expression, which prevents DSB repair and results in a significant increase in the number of cells in the S phase (Fig. 4). Furthermore, cells damaged and accumulated in the S and G2/M phases by HSA/ZnO-NP and radiation could not repair themselves. They were ultimately induced to undergo cell death (Fig. 5). This corresponds to a significant increase in apoptosis induction in the group treated with HSA/ZnO-NP combined with 2 or 4 Gy radiation compared to the control or radiation alone. Similarly, in the colony formation assay, the group treated with HSA/ZnO-NP combined with 2 or 4 Gy radiation had lower survival rates, at approximately 33.78% and 40.81%, respectively, compared to the group treated with radiation alone and finally, resulted in a 1.78-fold reduction in the radiation dose required for 50% HeLa cell viability when combined with HSA/ZnO-NP at 30 µg/mL. These results were consistent with previous research. I.C. da Costa Araldi et al. reported the radiosensitizing of cancer cells by treatment with resveratrol (RV) and its effect on the cell cycle. In their experiments with RV + IR on cancer cells, they observed changes in the cell cycle that caused an increase and arrest in S phases (da Costa Araldi et al. 2018). Moreover, Murali et al. (Murali et al. 2021) reported that ZnO nanoparticles can cause cell cycle arrest in the G2/M or S phases, which is directly related to the sensitivity of the cell line. Therefore, this study suggests that HSA/ZnO-NPs have the potential to be radiosensitizers, an effective strategy for treating cervical cancer. However, further studies must determine the mechanisms associated with cell death potentiated by combining HSA/ZnO-NPs and irradiation in the HeLa cell line.

Conclusion

The HSA/ZnO-NPs were successfully synthesized and characterized by SEM and DSL. These nanoparticles enhanced the cytotoxic effects of RT in a dose-dependent manner. The combination of HSA/ZnO-NPs with X-ray irradiation increased radiosensitivity by reducing the colony-formation ability and survival of HeLa cells, enhancing apoptosis induction, and causing cell cycle arrest, which may be linked to the activation of various cell death pathways. Consequently, this study suggests that HSA/ZnO-NPs could be promising nano-based radiosensitizers and an effective strategy for the treatment of cervical cancer.