Naringenin-Loaded Zinc Nitrate and Manganese Nitrate Nanoparticles Induce Caspase-Dependent Apoptosis in Kidney Cancer A-498 Cells

Abstract

Objective

Kidney cancer remains a major clinical concern due to its complex biology and resistance to conventional therapies. This study aimed to synthesize and characterize naringenin-loaded zinc nitrate and manganese nitrate nanoparticles (Naringenin-Zn/Mn NPs) and to evaluate their anticancer efficacy against A-498 kidney cancer cells.

Methods

The formulated nanoparticles were characterized using X-ray diffraction (XRD), UV–visible spectroscopy, energy-dispersive X-ray (EDX) analysis, scanning electron microscopy (SEM), Fourier transform infrared (FTIR) spectroscopy, photoluminescence (PL), and dynamic light scattering (DLS). Cytotoxicity was determined by MTT assay. Apoptosis was assessed using dual staining, while oxidative stress parameters and caspase enzyme activities were measured using standard biochemical assay kits.

Results

Characterization analyses confirmed the successful formation of crystalline, spherical Naringenin-Zn/Mn NPs with an average size of 135.90 nm and appropriate elemental composition. The nanoparticles significantly reduced A-498 cell viability in a dose-dependent manner (1-40 µg/ml). Treated cells showed elevated caspase activity, decreased antioxidant levels, and increased oxidative stress. Dual staining further demonstrated prominent apoptotic changes in nanoparticle-treated cells.

Conclusion

Naringenin-Zn/Mn NPs effectively inhibited proliferation and induced caspase-dependent apoptosis in A-498 kidney cancer cells. These findings suggest that the formulated nanoparticles may represent a promising nanotherapeutic strategy for kidney cancer management.

Introduction

Nanotechnology integrates multiple disciplines such as medicine, biology, physics, chemistry, and materials science by manipulating matter on a nanometric scale with the intention of designing materials with exceptional properties and functionalities. Such modification is possible via sophisticated and meticulous chemical and physical processes aimed at forming specific materials tailored for particular applications. ¹ Within the scope of nanotechnology, nanoparticles (NPs) have a pivotal role and are defined as particles with at least one dimension measuring less than 100 nanometers, having unique optical, thermal, electrical, chemical, and physical characteristics that differ greatly from those in bulk. The ability to manipulate materials at these dimensions has led to remarkable progress in medicine, chemistry, energy, and agriculture, among other fields. ² Another branch of nanotechnology, nanomedicine, applies nanoscaled materials and devices to medical purposes for exceptional advancements in diagnostics, drug delivery systems, treatment, and regenerative medicine. The more recent advances in nanotechnology have proved the usefulness of nanoscaled materials to electronics, catalysis, and biomedical research. ³

The development of nanobiotechnology focusing on biological processes demonstrates the cross-disciplinary integration within the field. That integration fosters the creation of complex nanodevices that aid in studying biological processes and biomedical developments at the molecular level, meeting the demands of clinical scientific research, medical practice, and preventive medicine. ⁴ The changing boundaries of various fields into biology and nanotechnology have created novel avenues for research. The remarkable advances of the past two decades include groundbreaking therapeutic research, including innovative cancer treatment systems. Targeted drug delivery systems. ³ These systems enhance drug effectiveness while diminishing undesirable effects and enabling tailored treatment approaches. The prospective strategies offered in the prevention and management of multiple diseases are broad because of the versatility of traditional techniques. The ability to design and engineer nanomaterials with precise on their functions opens a wide range of possibilities for diagnosing, treating, and preventing diseases, which improves the health of individuals. ⁵

Kidney cancer is one of the most complex types of malignancies, as it arises inside the renal parenchyma owing to the heterogeneous nature and multifaceted complexities associated with its pathogenesis. The most common type of kidney cancer is Renal Cell Carcinoma, which derives from the epithelium of the proximal convoluted tubule of the nephron, the kidney’s fundamental working component. ⁶ The incidence of kidney cancer is rising, particularly in developed nations, making it the seventh most frequently diagnosed malignancy. Annually, almost 270,000 new cases are identified globally, leading to around 116,000 fatalities. Renal cell carcinoma constitutes over 90% of all diagnosed kidney malignancies. ⁷ The etiology of kidney cancer is multifactorial, encompassing a complex interplay of genetic, environmental, and lifestyle causes. The pathophysiology of kidney cancer comprises a complex cascade of molecular mechanisms that interrupt typical cellular processes, leading to enhanced uncontrolled cell growth and survival. The insidious nature of kidney cancer often contributes to delayed diagnosis, as early-stage disease is frequently asymptomatic. This lack of early warning signs leads to a substantial proportion of patients presenting with metastatic disease, which poses a significant challenge to treatment and is a common group relatively resistant to radiation therapy and chemotherapy. ⁸

NPs are increasingly recognized for their crucial role in enhancing cancer therapies. The interaction of biological molecules with NPs, known as bioconjugation, further expands their utility in diagnostic and therapeutic applications. ⁹ An ideal NP-based agent should rapidly detect tumor cells, load multiple drugs, and enable real-time monitoring of efficacy. Nanotechnology integrates physical and chemical principles to create functional nanosized particles. ¹⁰ Phytochemicals, such as Naringenin (4, 5, 7-trihydroxyflavone), offer diverse pharmacological properties, including cardioprotective, anti-arthritic, anti-asthmatic, and anticancer effects.^11-14 While naringenin is abundant in citrus fruits, its therapeutic potential is often limited by poor solubility and systemic stability. Incorporating naringenin into NPs improves its bioavailability and targeted delivery, reducing side effects. ¹⁵ Despite the broad reporting of NP-based cell killing, a critical gap remains in the development of multi-functional, metal-based carriers tailored for specific malignancies like kidney cancer.

This study addresses this by synthesizing naringenin-loaded zinc nitrate (Zn) and manganese nitrate (Mn) NPs. Zinc is essential for cellular homeostasis and can induce oxidative stress in cancer cells, while Manganese offers unique potential for enhanced imaging and biochemical signaling. The present study illustrates the anticancer efficacy of naringenin-loaded zinc nitrate (Zn–Nar) and manganese nitrate (Mn–Nar) nanoparticles against human kidney cancer A-498 cells (Figure 1). The novelty of this approach lies in the synergistic combination of these two distinct metal ions with naringenin to create a dual-action delivery system. As there is limited literature on naringenin’s efficacy against renal malignancies, this work aims to characterize these Naringenin-Zn/Mn NPs and investigate their innovative potential in inducing apoptosis and reducing viability in A-498 kidney cancer cells.

Figure 1.

Schematic representation of the anticancer mechanism of naringenin-loaded zinc nitrate (Zn–Nar) and manganese nitrate (Mn–Nar) nanoparticles in human kidney cancer A-498 cells

Materials and Methods

Chemicals

The major chemicals utilized in this study, including naringenin, Zn, and Mn, were acquired from Sigma-Aldrich, USA. The standard diagnostic kits for biochemical analyses were obtained from Elabscience, USA.

Synthesis of Naringenin-Zn/Mn NPs

Naringenin-Zn/Mn NPs were synthesized by dissolving 0.1 M Zinc nitrate and 0.1 M Manganese (II) nitrate hexahydrate (Mn(NO3)2·6H2O) in 100 mL of distilled water. Following this, 50 mg of naringenin was added to the metal solution. To initiate the precipitation of the nanoparticles, 0.1 M NaOH was added dropwise under continuous magnetic stirring until the solution reached an alkaline pH (approximately pH 9–10). The mixture was then heated at 80 °C for four hours; during this period, the incremental change in pH and thermal energy triggered the formation of a distinct white precipitate. Once the reaction was complete, the resulting precipitate was collected, washed, and desiccated at 100 °C to remove residual moisture. Finally, the Zn/Mn nanopowder was annealed at 600 °C for 5 hours to achieve the desired crystalline structure.

Characterization of the Synthesized Naringenin-Zn/Mn NPs

The X-ray diffraction (XRD) patterns of the synthesized Naringenin-Zn/Mn NPs were obtained utilizing an XRD-600 Diffractometer (Shimadzu, Japan) at 30 kV and 30 mA. The scanning rate was established at 2°/min, and the XRD measurements were conducted within the 2–60° range of 2θ angles.

The Naringenin-Zn/Mn NPs sample was studied using a scanning electron microscope (SEM; FEI, USA). A 5 μl aliquot of the sample was layered onto an aluminum stub and subsequently dried for 24 hours at 37°C. Elemental analysis of the Naringenin-Zn/Mn NPs was accomplished with an EDX spectrometer integrated with the SEM.

The Naringenin-Zn/Mn NP’s morphology was studied using a PHILIPS CM300 (USA) transmission electron microscope (TEM). The copper grid, which holds the Naringenin-Zn/Mn NPs sample, was put into an electromagnetic field in a vacuum. Microphotographs of the specimen have been achieved using the scanning electron beam.

The distribution patterns and the mean particle size of the Naringenin-Zn/Mn NPs were measured using a DLS instrument (HORIBA, Kyoto, Japan). The functional groups associated with the synthesized NPs were studied with Fourier transform infrared spectroscopy (FTIR) (Thermo Nicolet-6700, ThermoScientific, USA. The NPs were studied with the JASCO770 UV-Vis spectrophotometer (Tokyo, Japan) to acquire their UV-visible spectrum. The photoluminescence (PL) spectra of NPs were acquired with a Perkin Elmer-LS 14 spectrometer (Perkin Elmer, USA).

In Vitro Assays

Collection of Cell Culture

The A-498 kidney cancer cells were acquired from ATCC, USA. Then, cells were cultivated in DMEM enriched with FBS (10%) in a CO2 incubator at 37°C. Cells were trypsinized and utilized for subsequent experiments upon achieving 80% confluency.

MTT Assay

The MTT assay was employed to evaluate the viability of control and NPs-treated A-498 cells. Cells were cultivated in a 96-well plate at 5 × 10³ cells/well population for 24 h. After incubation, the cells were treated with different concentrations (1, 2.5, 5, 7.5, 10, 20, and 40 µg/ml) of the Naringenin-Zn/Mn NPs for 24 hours. Later, 20 µl of MTT reagent and 100 µl of DMEM were added to each well for 4 hours. The formazan deposits developed in the wells were subsequently liquefied with DMSO (100 µl), and the absorbance was detected at 570 nm.

Dual Staining

Apoptotic incidences in the control and Naringenin-Zn/Mn NPs-treated A-498 cells were examined using a dual staining experiment. A-498 cells were cultivated in a 24-well plate and exposed to 7.5 and 10 μg/ml of the synthesized Naringenin-Zn/Mn NPs for 24 hours. Subsequently, a 100 μg/ml solution of AO/EB dye mixture was added to the wells for a duration of 5 minutes in a dark environment. The stained cells were examined with a fluorescence microscope to identify the occurrence of apoptosis.

Analysis of Oxidative Stress Indicators and Apoptotic Proteins

The amounts of oxidative stress biomarkers malondialdehyde (MDA), superoxide dismutase (SOD), glutathione (GSH), and catalase (CAT) in cell lysates of A-498 cells treated with NPs and control cells were determined using commercial assay kits from Elabscience, USA. The caspase-3, -8, and -9 concentrations in the cell lysates of control and NPs-exposed A-498 cells were studied using diagnostic kits (MyBioSource, USA). Each test was done in triplicate using the specifications given by the manufacturer.

Statistical Analysis

The statistical studies were done using GraphPad Prism, and the values are illustrated as the mean±SD of triplicates. The values were examined using one-way ANOVA and Tukey’s post hoc, with p<0.05 designated as significant.

Results

Characterization Results of the Naringenin-Zn/Mn NPs

The XRD analysis was employed to assess the purity and crystalline properties of the synthesized Naringenin-Zn/Mn NPs (Figure 2A). The distinct peaks illustrated in Figure 2A, including (100), (002), (101), (121), (102), (441), (110), (103), (200), (112), and (201), denote the exact crystallographic orientations of the synthesized Naringenin-Zn/Mn NPs in their crystalline state.

Figure 2.

XRD and DLS analysis of the synthesized Naringenin-Zn/Mn NPs. The distinct peaks observed in the XRD result denote the crystallographic orientations of the synthesized Naringenin-Zn/Mn NPs in their crystalline state (A). The DLS results indicate a tiny agglomeration with an average hydrodynamic diameter of 135.90 nm (B)

The DLS was executed to evaluate the hydrodynamic size of the synthesized Naringenin-Zn/Mn NPs, as illustrated in Figure 2B. The investigation indicates a tiny agglomeration with an average hydrodynamic diameter of 135.90 nm.

The shape of the synthesized Naringenin-Zn/Mn NPs was examined via SEM analysis, as depicted in Figure 3A. The images at lower and higher magnifications demonstrate that the Naringenin-Zn/Mn NPs have spherical structures with a certain level of aggregation. These observations validate the effective coating of Mn and naringenin onto the Zn surface matrix. Figure 3B displays an EDAX spectrum of the synthesized Naringenin-Zn/Mn NPs. The principal constituents discovered were oxygen (O), carbon (C), Zinc (Zn), and manganese (Mn), thereby affirming their existence in the Naringenin-Zn/Mn NPs. The EDAX analysis further corroborates the synthesis of the Naringenin-Zn/Mn NPs.

Figure 3.

SEM and EDAX analysis of the synthesized Naringenin-Zn/Mn NPs. The SEM images illustrate that the Naringenin-Zn/Mn NPs have spherical structures with a certain level of aggregation (A). EDAX spectrum of the synthesized Naringenin-Zn/Mn NPs demonstrated the presence of oxygen (O), carbon (C), Zinc (Zn), manganese (Mn) (B)

Figure 4A–E presents the microphotographs of the produced Naringenin-Zn/Mn NPs, obtained from TEM examination. The TEM microphotographs indicated that the produced Naringenin-Zn/Mn NPs exhibited spherical forms with irregular distributions. The analysis of the SAED patterns (Figure 4F) validated the crystallization of the Naringenin-Zn/Mn NPs.

Figure 4.

TEM analysis of the synthesized Naringenin-Zn/Mn NPs. The TEM microphotographs indicated that the Naringenin-Zn/Mn NPs exhibited spherical forms with irregular distributions (A-E). The SAED patterns validated the crystallization of the Naringenin-Zn/Mn NPs (F)

The FT-IR was conducted to identify the surface functional groups associated with the synthesized Naringenin-Zn/Mn NPs (Figure 5A). The produced Naringenin-Zn/Mn NPs exhibited several peaks distributed across various frequencies. The notable peaks at 3288 cm-1, 3110 cm-1, and 3031 cm-1 correspond to the stretching motion of the O-H bond. The hydroxyl stretching is shown by several peaks observed at wavenumbers of 2917 cm-1, 2824 cm-1, 2705 cm-1, and 2626 cm-1. The peaks at wavenumbers 1899 cm-1, 1638 cm-1, 1602 cm-1, 1518 cm-1, 1464 cm-1, 1390 cm-1, 1311 cm-1, and 1247 cm-1 are ascribed to the bending vibrations of C-H and C-O bonds. The peaks observed at 1153 to 431 cm-1 signify the occurrence of H-O bonds.

Figure 5.

FT-IR, UV-vis spectroscopy, and PL analyses of the synthesized Naringenin-Zn/Mn NPs. The FT-IR analysis results demonstrated the occurrence of numerous surface functional groups in the synthesized Naringenin-Zn/Mn NPs (A). The Naringenin-Zn/Mn NPs development was validated using UV-visible spectrophotometric analysis, with the highest peaks at 323 and 376 nm (B). The excitations of the Naringenin-Zn/Mn NPs were recorded at several wavelengths. The PL spectrum elucidates the crystal structure, surface characteristics, and structural defects of Naringenin-Zn/Mn NPs (C)

The formation of Naringenin-Zn/Mn NPs was validated using UV-visible spectrophotometric analysis. The absorbance was measured from 200 to 1000 nm, indicating the highest peaks at 323 and 376 nm, which confirms the presence of Naringenin-Zn/Mn NPs (Figure 5B).

Figure 5C illustrates the results of a PL study conducted on the synthesized Naringenin-Zn/Mn NPs. The precise wavelengths corresponding to the excitations of the Naringenin-Zn/Mn NPs were 360, 383, 397, 404, 412, 432, 453, 474, 489, and 497 nm, respectively. The PL spectrum elucidates information regarding the crystal structure, surface characteristics, and structural defects illustrated by the Naringenin-Zn/Mn NPs. The observation of peaks at wavelengths of 360, 383, and 397 nm signifies the occurrence of free exciton recombination. Interstitial oxygen vacancies account for the green emission detected at the 404, 412, 432, and 453 nm peaks. The observation of green emission at precise wavelengths at 474, 489, and 497 nm signifies the existence of a singly ionized oxygen vacancy in the synthesized Naringenin-Zn/Mn NPs, as illustrated in Figure 5C.

Effect of Naringenin-Zn/Mn NPs on the Growth of A-498 Cells

The MTT cytotoxicity experiment was conducted to evaluate the cytotoxicity of the Naringenin-Zn/Mn NPs on A-498 cells. The results are depicted in Figure 6. The results indicate that the administration of Naringenin-Zn/Mn NPs at several doses (1-40 μg/ml) significantly reduced the viability of A-498 cells. Cell viability showed a marked reduction at elevated concentrations of the Naringenin-Zn/Mn NPs in comparison to untreated cells. The A-498 cells exhibited greater sensitivity to the Naringenin-Zn/Mn NPs treatment, and the IC50 concentration was fixed to be 7.5 μg/ml, which was selected for further studies.

Figure 6.

Effect of Naringenin-Zn/Mn NPs on the viability of A-498 cells. The values are statistically evaluated using Graphpad Prism, and results are depicted as a mean±SD of triplicates. The results are studied using a one-way ANOVA and Tukey’s post hoc test to ascertain the significance. Values of the treatment groups differ significantly at p<0.05 from the control group

Effect of Naringenin-Zn/Mn NPs on the Apoptosis in A-498 Cells

Figure 7 illustrates the results of the dual staining technique, which validates the apoptotic activity of the synthesized Naringenin-Zn/Mn NPs. The results reveal that treatment with Naringenin-Zn/Mn NPs at 7.5 and 10 µg/ml doses, respectively, resulted in an increased quantity of yellow/red fluorescent cells relative to the control. The green fluorescent cells in the control group denote live cells, while the elevated quantity of yellow/red fluoresced cells in Naringenin-Zn/Mn NPs-treated A-498 cells implies the onset of apoptosis (Figure 6).

Figure 7.

Effect of Naringenin-Zn/Mn NPs on the apoptosis in A-498 cells. The green fluoresced cells in the control group indicate the presence of viable cells, while the elevated quantity of yellow/red fluoresced cells in the Naringenin-Zn/Mn NPs-treated cells denotes the onset of apoptosis

Effect of Naringenin-Zn/Mn NPs on the Oxidative Stress Markers in the A-498 Cells

Figure 8 depicts the effect of Naringenin-Zn/Mn NPs treatment on the concentrations of oxidative stress-related markers in the A498 cells. The treatment with the Naringenin-Zn/Mn NPs demonstrated increased MDA levels. Furthermore, the NPs treatment considerably diminished the GSH, CAT, and SOD concentrations in A-498 cells. These results indicated the decrease in antioxidant levels alongside an increase in oxidative stress in the Naringenin-Zn/Mn NPs-treated A-498 cells.

Figure 8.

Effect of Naringenin-Zn/Mn NPs on the oxidative stress markers in the A-498 cells. The values are statistically evaluated using Graphpad Prism, and results are depicted as a mean±SD of triplicates. The results are studied using a one-way ANOVA and Tukey’s post hoc test to ascertain the significance. Values of the treatment groups differ significantly at p<0.05 from the control group

Effects of Naringenin-Zn/Mn NPs on Caspase Activities in A-498 Cells

The influence of the formulated Naringenin-Zn/Mn NPs on the caspase activities in A-498 cells was studied, with findings illustrated in Figure 9. The administration of the formulated NPs led to an elevated caspase-3, -8, and -9 activities in A-498 cells in comparison with control cells. These outcomes suggest that the Naringenin-Zn/Mn NPs may trigger apoptosis in A-498 cells via augmenting caspases.

Figure 9.

Effect of Naringenin-Zn/Mn NPs on the caspase enzyme activities in the A-498 cells. The values are statistically evaluated using Graphpad Prism, and results are depicted as a mean±SD of triplicates. The results are studied using a one-way ANOVA and Tukey’s post hoc test to ascertain the significance. Values of the treatment groups differ significantly at p<0.05 from the control group

Discussion

Contemporary cancer treatment modalities, such as chemotherapy, surgery, and radiotherapy, are constrained by systemic toxicity and lack of selectivity. The development of drug resistance during treatment is particularly challenging and requires new approaches to therapy. ¹⁶ The shortcomings of such standard strategies may result in sub-therapeutic concentrations of the therapeutic agent being delivered to the tumor, which, unfortunately, impacts surrounding healthy cells and contributes toward severe side effects, thus undermining the overall therapy efficacy. ¹⁷ Recently, the advancement of nanotechnology and its related domains, such as NP systems and nanomedicine, has emerged as an effective solution specifically targeted at addressing these challenges. With their unique size-dependent attributes, large surface area to volume ratio, and ease of surface changes, NPs can be designed to selectively aim at cancer cells, optimize drug delivery, and lower non-target effects. Nanodrug systems allow for focused drug distribution, which increases drug accumulation at the tumor while reducing overall body impact, thus making them feasible and promising prospects for use in cancer treatment. ¹⁸

NP-based drug delivery systems have demonstrated potential in cancer treatment and management through advantageous pharmacokinetics, accurate targeting abilities, reduced side effects, and alleviation of drug resistance. Merging nanomaterials with cancer treatment is an evolving domain with the capability to change the fight against cancer. In terms of NP usage, targeted drug delivery’s main benefit is the fewer doses and side effects suffered by cancer patients. ¹⁹ The cytotoxic effect of NPs is augmented when NPs are internalized, thus making NP’s internal modifications with targeted ligands effective for drug delivery. Such modifications permit NPs as drug carriers to efficiently encapsulate the drugs, protecting them against degradation while in circulation, enhancing their solubility, and mitigating their toxic side effects. NPs possess adjustable pharmacokinetic and pharmacodynamic properties that can be optimized for better efficacy and the drug’s effectiveness. NPs altered with targeting moieties can be recognized and bound to specific receptors on the surface of cancer cells, where they are internalized via receptor-mediated endocytosis, leading to controlled release of cytotoxic agents, which kill the cell from inside. ²⁰

The size, shape, surface charge, and composition of NPs have determining impacts on their behaviors, circulation time, biodistribution, and cellular uptake. Physicochemical characterization of synthesized NPs is arguably most critical due to the intricate relationship between their physicochemical properties and their performance across a wide spectrum of applications. Tailored NP synthesis requires understanding the specific attributes that differentiate these materials, and relevant NP characterization is essential. ²¹ Characterization contributes to knowledge of NP size, shape, surface charge, crystallinity, composition, stability, and all essential features that determine the function of the NPs. Comprehensive methodologies also serve to maintain reproducibility and scalability of the NP synthesis methods. Enhanced use of NPs in various regions highlights the need to understand their possible health effects, which requires thorough characterization. ²² In this study, the UV-visible spectroscopic data showing absorbance at peaks 323 nm and 376 nm confirmed the formation of Naringenin-Zn/Mn NPs. The XRD results confirmed the crystallographic orientations of the synthesized Naringenin-Zn/Mn NPs in crystalline form. Images captured by SEM and TEM, respectively, clearly show Naringenin-Zn/Mn NPs to be spherical in shape. However, certain portions displayed some degree of clustering. The EDAX analysis provided evidence for the presence of many elements such as oxygen (O), carbon (C), zinc (Zn), and manganese (Mn) within the NPs. Based on DLS analysis, lower levels of clustering, or agglomeration, were recorded, with an average hydrodynamic diameter of 135.90 nm. Various stretching and bonding modes of vibrations in the Naringenin-Zn/Mn NPs were confirmed by FT-IR results. Crucially, the interaction between these metallic NPs and ionizing radiation, such as that used in XRD or EDX characterization, provides insight into their potential as radiosensitizers. It has been reported that irradiated metallic NPs can emit secondary electrons and generate ROS, significantly increasing their therapeutic efficacy. ²³ In the context of our Zn/Mn-based system, this suggests that the metallic core does not merely act as a carrier for Naringenin but may also enhance cancer cell killing through radiation-induced oxidative stress. This synergy between the phytochemical payload and the radiation-sensitive metallic components highlights the innovative potential of Naringenin-Zn/Mn NPs as multi-modal agents in advanced cancer therapy.

Apoptosis removes unwanted or damaged cells and prevents tumorigenesis to help maintain tissue homeostasis. In tumor cells, however, certain disruptions to this process, called apoptosis, tend to occur, enabling uncontrolled proliferation and evasion of standard treatment. ²⁴ There are various cancer treatments available today, and most utilize the induction of apoptosis as a primary mechanism of action. Resistance mechanisms in tumor cells still need to be better understood to develop therapies that would selectively eliminate malignant cells, rendering them ineffective. ²⁵ Cancer as a disease is marked by evasion of apoptosis, which in itself is a hallmark feature, underscoring the cells’ ability to have DNA damage with oncogenic mutations and proliferate without control. ²⁶ Dysregulation in the apoptotic pathway may occur at multiple junctures, such as through the downregulation of tumor suppressor genes that typically facilitate apoptosis in response to cellular stress. ²⁷ The apoptotic evasion often involves the overexpression of anti-apoptotic proteins like Bcl-2, which arrest caspase activation and shift the delicate equilibrium of cell survival. ²⁸ In this study, dual staining confirmed that Naringenin-Zn/Mn NPs effectively induce apoptosis in A-498 kidney cancer cells. Notably, this apoptotic effect followed a dose-dependent relationship, a common hallmark of potent nano-therapeutics. The mechanism behind this relationship likely stems from the dose-proportional internalisation of the nanoparticles, which triggers a surge in intracellular reactive oxygen species (ROS) and subsequent mitochondrial dysfunction. As highlighted in recent advances in nanomaterial therapy, the controlled release of metallic ions and bioactive payloads (like Naringenin) can bypass traditional resistance mechanisms by directly targeting the mitochondrial membrane potential or inhibiting DNA repair enzymes.^29,30 At higher concentrations, the Naringenin-Zn/Mn NPs likely saturate the cancer cell’s antioxidant defenses, forcing the transition from pro-survival signaling to programmed cell death. These findings suggest that Naringenin-Zn/Mn NPs inhibit kidney cancer progression by restoring the sensitivity of A-498 cells to apoptotic triggers through a synergistic, concentration-sensitive delivery system.

Oxidative stress, defined by an imbalance between the ROS generation and the ability of the cellular antioxidant systems to neutralize them, plays a multifaceted role in the onset of cancer. The disruption of equilibrium between oxidants and antioxidants leads to oxidative damage affecting major cellular components, potentially triggering apoptosis. ³¹ ROS can directly induce apoptosis through oxidation of cellular components or indirectly activate apoptotic pathways. The consequences of oxidative stress can be far-reaching, affecting the overall health status through lipid peroxidation, physiological perturbations, and alterations in metabolic pathways. Due to rapid proliferation coupled with limited nutrient supply in the tumor microenvironment, cancer cells are subjected to metabolic stress and can potentially undergo cell death via ROS-induced apoptosis. ³² The application of oxidative stress to tumor cells presents a viable option for treatment. Tumor cells usually have high levels of ROS relative to non-malignant cells, hence they are more susceptible to additional oxidative damage. It has been reported that ROS level-elevating agents or antioxidant suppressors would cause selective apoptosis in these cancer cells. ³³ The current work’s findings emphasize that treatment of A-498 cells with NPs has markedly increased oxidative stress due to the depletion of antioxidants. Cells treated with formulated NPs appear to possess high levels of oxidative stress, which may aid in apoptosis. Thus, it was clear that Naringenin-Zn/Mn NPs can trigger apoptosis through oxidative stress mechanisms in kidney cancer cells.

Apoptotic pathways are intricate, involving a sequence of molecular events directed by caspases, a family of cysteine proteases that act as the executors of apoptosis. The caspase family modulates cell death by accurately cleaving several intracellular protein targets. An apoptosis executioner, Caspase-3, is activated through both intrinsic and extrinsic pathways, which mark the final stages of apoptosis. This type of activation starts the irreversible destruction of a cell by removing the central parts of the cell, while also causing shape alterations associated with apoptosis. ³⁴ Activated caspase-3 cleaves several substrates, including structural proteins like actin and lamin, resulting in cytoskeletal disintegration and nuclear fragmentation. Also, DNA repair enzymes undergo inactivation by caspase-3, which further weakens the ability of the cell to repair the damaged DNA and enhances apoptosis. Activation of caspase-3 is one of the most crucial steps in the apoptosis process, as it starts the entire cascade of events that leads to the disassembly of the cell. ³⁵

Caspase-8 is an initiator caspase that is mostly linked with the extrinsic apoptotic pathway, which is activated by the cell membrane death receptors. Death receptors trigger apoptosis upon interaction with their corresponding ligands, such as Fas ligand. Upon ligand engagement, these receptors recruit adaptor proteins, such as FADD, forming a death-inducing signaling complex. This combination subsequently activates caspase-8, initiating the caspase pathway. Caspase-8 directly activates downstream executioner caspases, such as caspase-3, leading to cellular disassembly. Caspase-8 can degrade Bid, a pro-apoptotic protein, which then translocates to the mitochondria and initiates the intrinsic apoptotic cascade. The extrinsic pathway, activated by caspase-8, offers a direct mechanism for apoptosis, circumventing mitochondrial participation in specific scenarios. ³⁶

Caspase-9 is an initiator caspase operating within intrinsic apoptotic pathways. It gets activated by intracellular signals such as oxidative stress, DNA damage, and insufficient growth factors. These stress signals lead to the mitochondria releasing cytochrome c into the cytoplasm. Cytochrome c subsequently associates with Apaf-1, resulting in the formation of the apoptosome, a multi-protein complex that activates caspase-9. Upon activation, caspase-9 subsequently activates downstream executioner caspases, including caspase-3, ultimately resulting in cellular death. ³⁷ The simultaneous activation of caspase-3, -8, and -9 is necessary for certain types of cancer cells to undergo apoptosis. The relationship between caspases is intricate due to the interplay between the extrinsic and intrinsic pathways, which furthers the apoptotic cascade. For the design of effective therapies that selectively trigger apoptosis in neoplastic cells, comprehensive knowledge of these pathways, including the roles of these caspases and the controllers of their activation thresholds, is essential. ³⁸ This study illustrates that the Naringenin-Zn/Mn NPs treatment significantly enhanced the activities of caspase-3, -8, and -9 in A-498 kidney cancer cells. The elevated caspase activity in cells subjected to Naringenin-Zn/Mn NPs may promote apoptosis. The Naringenin-Zn/Mn NPs treatment was demonstrated to trigger caspase-dependent apoptosis in A-498 kidney cancer cells.

The present research work has some considerable limitations, such as the in vitro nature of the research, which limits direct extrapolation to in vivo systems. The study focused on A-498 kidney cancer cells, necessitating further research across diverse cell lines and cancer types. Mechanistic insights into nanoparticle-cell interactions remain incomplete, warranting deeper investigation. Long-term cytotoxicity and biocompatibility of Naringenin-Zn/Mn NPs in normal cells require additional study. The lack of in vivo pharmacokinetics, biodistribution, and tumor-targeting data restricts clinical translation understanding. Optimization of nanoparticle dosing and administration routes is needed. Further work is required to explore potential synergies with existing therapies. Effects on non-target organs and tissues remain uncharacterized. Scale-up and stability aspects for clinical use need addressing. Overall, while promising, this study’s findings require validation through expanded in vitro and in vivo studies. Future studies are needed to address these limitations and advance Naringenin-Zn/Mn NPs toward clinical application.

Conclusion

The current study highlights that the Naringenin-Zn/Mn NPs markedly decrease growth and promote apoptosis in A-498 cells. The Naringenin-Zn/Mn NPs have also been effective in decreasing cell survival, increasing oxidative stress, and initiating apoptosis through caspase pathways in A-498 cells. The findings indicate that Naringenin-Zn/Mn NPs may serve as a potent therapeutic agent for the treatment of kidney cancer. Additional investigations are necessary to elucidate the distinct molecular pathways regulating the anti-cancer effects of Naringenin-Zn/Mn NPs against kidney cancer.