Phyto-Mediated Green Synthesis of Titanium Dioxide Nanoparticles Using Punica granatum Peel Extract with Anticancer Activity Against A۵۴۹ Lung Carcinoma Cells

Titanium dioxide nanoparticles (TiO۲ NPs) have attracted increasing attention in nanobiotechnology due to their unique photocatalytic, optical, and surface properties, which enable applications in cancer therapy, drug delivery, and antimicrobial coatings. However, conventional physicochemical synthesis routes often require harsh conditions, toxic reducing agents, and post-synthesis treatments such as high-temperature calcination, which can introduce environmental and biological safety concerns. In recent years, green synthesis approaches based on plant extracts have emerged as an eco-friendly alternative for nanoparticle production. Phytochemicals such as polyphenols, flavonoids, and tannins simultaneously act as reducing, capping, and stabilizing agents, enabling the formation of nanoscale structures under mild conditions. Pomegranate peel is particularly rich in phenolic compounds and exhibits inherent antioxidant and anticancer properties, making it a promising candidate for the biofabrication of metal oxide nanoparticles. Considering the global burden of lung cancer and the importance of safer nanomaterials for biomedical use, there is a growing need to develop biocompatible TiO۲ NPs with controlled size, colloidal stability, and selective cytotoxicity against cancer cells. Objective The aim of this study was to biosynthesize TiO۲ nanoparticles via a green route using pomegranate peel extract, characterize their physicochemical properties by dynamic light scattering (DLS), transmission electron microscopy (TEM), and zeta potential analysis, and evaluate their cytotoxic and antiproliferative effects on human lung carcinoma A۵۴۹ cells using the MTT assay. Materials and Methods Green synthesis of TiO۲ nanoparticles Dried pomegranate peel powder was used as a natural source of phytochemicals. An aqueous extract was prepared under mild heating and filtration to obtain a clear, phenolic-rich solution. This extract was then mixed with a titanium precursor (titanium tetraisopropoxide, TTIP) under controlled stirring and temperature. The phytochemicals present in the extract functioned as bioreducing and capping agents, facilitating the formation of TiO۲ nanoparticles without the use of additional toxic chemicals or high-temperature calcination. The resulting colloidal dispersion was separated by centrifugation, washed with deionized water and ethanol to remove unreacted components, and gently dried at moderate temperature to yield a stable TiO۲ nanoparticle powder. Physicochemical characterization The hydrodynamic diameter and size distribution of the biosynthesized TiO۲ NPs were determined by dynamic light scattering (DLS). The surface charge and colloidal stability were evaluated using zeta potential measurements. Morphology and primary particle size were assessed by transmission electron microscopy (TEM). According to the patent-based data, TEM micrographs showed predominantly quasi-spherical nanoparticles with a size range of approximately ۲۰-۸۰ nm, while DLS analysis indicated a slightly larger hydrodynamic diameter in the range of ۸۰-۹۰ nm, with an average size around ۸۷ nm, reflecting the contribution of the phytochemical corona and solvation layer. The measured zeta potential was approximately +۳۱ mV, indicating a positively charged surface and relatively good colloidal stability. Cell culture and MTT assay on A۵۴۹ cells Human lung carcinoma A۵۴۹ cells were cultured under standard conditions in appropriate culture medium supplemented with fetal bovine serum and antibiotics. For cytotoxicity evaluation, cells were seeded into ۹۶-well plates and allowed to attach overnight. Biosynthesized TiO۲ NPs were freshly dispersed in culture medium and applied to A۵۴۹ cells at different concentrations. After a defined exposure period, cell viability was assessed using the MTT assay. Absorbance was measured at ۵۷۰ nm, and cell viability was expressed as a percentage relative to untreated controls. Dose-dependent cytotoxicity curves were plotted and the IC۵۰ value was estimated from the viability data. Results TEM and DLS analysis TEM imaging confirmed the successful green synthesis of TiO۲ nanoparticles using pomegranate peel extract. The nanoparticles exhibited predominantly spherical to quasi-spherical morphology with primary particle sizes in the range of ۲۰-۸۰ nm, indicating that the phytochemical matrix effectively controlled the nucleation and growth of TiO۲ NPs under mild conditions. No large micrometer-scale aggregates were observed in the micrographs, suggesting a relatively uniform nanoscale morphology. DLS measurements revealed a hydrodynamic diameter in the range of ۸۰-۹۰ nm, with a mean size of approximately ۸۷ nm. The observed difference between TEM and DLS measurements is consistent with the presence of a hydration shell and the organic capping layer derived from the plant extract, which increase the effective hydrodynamic size in suspension. The size distribution was relatively narrow, indicating a reasonably monodisperse population suitable for biological applications. Zeta potential and colloidal stability Zeta potential analysis showed that the biosynthesized TiO۲ NPs possessed a positive surface charge of about +۳۱ mV. This magnitude of zeta potential suggests moderate to good electrostatic stabilization of the colloidal system, reducing the likelihood of rapid aggregation in aqueous media. The positive surface charge can facilitate interactions with negatively charged cellular membranes, potentially enhancing cellular uptake and internalization in lung cancer cells. Cytotoxicity against A۵۴۹ cells (MTT assay) MTT assay results demonstrated that the green-synthesized TiO۲ NPs exerted a dose-dependent cytotoxic effect on A۵۴۹ cells. At lower concentrations, a partial reduction in cell viability was observed, while at higher concentrations near and above ۴۰ μg/mL, a marked decrease in metabolic activity was detected, reflecting substantial inhibition of cell proliferation. Based on the dose-response profile derived from the patent data, the IC۵۰ value was estimated around the vicinity of this concentration range, indicating significant antiproliferative activity against A۵۴۹ cells. Although detailed apoptosis/necrosis profiling was not included in the present dataset, the marked reduction in viability at relatively low micromolar-equivalent doses suggests that the biosynthesized TiO۲ NPs possess a pronounced cytotoxic potential toward lung carcinoma cells. These effects are likely mediated by reactive oxygen species (ROS) generation, mitochondrial dysfunction, and membrane damage, as commonly reported for metal oxide nanoparticles, in combination with the intrinsic bioactivity of pomegranate-derived surface moieties. Conclusion In this study, TiO۲ nanoparticles were successfully produced via a green synthesis route using pomegranate peel extract as a natural reducing, capping, and stabilizing agent, avoiding harsh synthetic conditions and high-temperature calcination. TEM and DLS analyses confirmed the formation of nanosized particles with a primary size range of ۲۰-۸۰ nm and a hydrodynamic diameter around ۸۷ nm, while a zeta potential of approximately +۳۱ mV indicated favorable colloidal stability and a positively charged surface. The biosynthesized TiO۲ NPs exhibited significant, dose-dependent cytotoxicity against human lung carcinoma A۵۴۹ cells in the MTT assay, with an IC۵۰ in the tens of μg/mL range, highlighting their potential as anticancer nanomaterials. The combination of eco-friendly synthesis, controlled nanoscale dimensions, and pronounced antiproliferative effects suggests that green TiO۲ NPs derived from pomegranate peel could be promising candidates for further development in lung cancer nanotherapy and related biomedical applications. Future studies should focus on mechanistic analyses of cell death pathways (apoptosis versus necrosis), evaluation of selectivity toward cancer versus normal cells, and surface functionalization strategies to enhance tumor targeting while minimizing off-target toxicity.

کلیدواژه ها:

TiO۲ NPs ، Green Synthesis ، Pomegranate Peel Extract ، A۵۴۹ Lung Carcinoma Cells