Niosome-Encapsulated Selenium Nanoparticles: A Novel Paradigm for Cancer Therapy

Abstract

Overview: This study explores a novel therapeutic method for oral squamous cell carcinoma using selenium nanoparticles encapsulated within niosomal carriers, targeting enhanced stability, cellular uptake, and controlled drug release.

Methods: Selenium nanoparticles were encapsulated into niosomes via thin-film hydration and characterized by dynamic light scattering (DLS) for particle size, zeta potential, and polydispersity index (PDI). Cytotoxicity was assessed using the MTT assay on CAL27 cells after 24 hours, alongside a drug release study conducted under simulated physiological conditions.

Results: The formulated nanoparticles showed an average size of ~180 nm, zeta potential of –25 mV, and low PDI (~0.15), indicating high stability and uniformity. MTT results indicated a 60% decrease in cell viability compared to controls after 24 hours. The drug release exhibited an initial burst (35% release in 6 hours), followed by sustained release reaching ~90% over 48 hours.

Conclusion: Niosome-encapsulated selenium nanoparticles display promising physicochemical characteristics, significant cytotoxic activity, and a controlled release profile, underscoring their potential as an effective therapeutic approach for oral squamous cell carcinoma. Further in vivo studies are recommended to validate clinical applicability.

Introduction

The digitalization of healthcare and technology-driven research have provided valuable insights into the impact of digital addiction on mental health, highlighting the need for innovative intervention strategies [1]. Technological advancements have significantly contributed to the treatment of various diseases, revolutionizing modern medicine and improving patient outcomes [2-7]. Here are several notable examples of technological advancements across various fields. Technological advancements in supply chain optimization have played a crucial role in ensuring the resilience of pharmaceutical distribution during pandemics, improving patient care and access to essential medications [8]. Advances in genetic engineering have unlocked new possibilities for immunotherapy, improving the therapeutic potential of CAR-NK cell therapy for cancer treatment [9]. Innovations in material science, such as sulfur-driven reactive processing, have led to the development of high-performance biomedical composites with potential applications in medical implants and prosthetics [10]. Advances in dental biomaterials, such as casein phosphopeptide-amorphous calcium phosphate formulations, have improved remineralization potential and bonding strength, enhancing restorative dentistry outcomes [11]. The integration of clinical data with pathological findings has improved the accuracy of disease diagnosis, leading to more precise and personalized treatment plans in oral healthcare [12]. Advances in biomedical research have enabled the development of novel therapeutic approaches, such as synbiotics, to improve metabolic disorders and inflammatory conditions in patients with non-alcoholic fatty liver disease [13]. Cutting-edge molecular research has revealed the role of gene regulation in cancer progression, paving the way for targeted therapies in conditions such as papillary thyroid carcinoma [14]. The integration of advanced diagnostic techniques in prenatal care has significantly enhanced the accuracy of detecting fetal abnormalities, improving maternal and neonatal health outcomes [15]. Oral squamous cell carcinoma (OSCC) remains a formidable global health burden, characterized by its aggressive nature, high recurrence rates, and resistance to conventional therapeutic modalities [16- 17]. Environmental and lifestyle factors play a crucial role in disease development, emphasizing the need for preventive strategies and public health policies to reduce the incidence of oral cancer [18]. Current treatment strategies, including chemotherapy, often result in significant off-target cytotoxicity, suboptimal drug bioavailability, and multidrug resistance, thereby necessitating the development of innovative and precisely targeted therapeutic interventions [19-20]. The application of combination therapies utilizing chemotherapeutic agents and natural compounds like curcumin has demonstrated significant potential in overcoming drug resistance in oral cancer treatment [21]. The emergence of nanotechnology-driven drug delivery platforms has revolutionized cancer therapy, offering enhanced pharmacokinetics, targeted drug delivery, and controlled release mechanisms that mitigate systemic toxicities and improve therapeutic efficacy [22]. The emergence of nanotechnology in drug delivery has revolutionized cancer treatment by enabling targeted therapies, such as folic acid-conjugated nanoniosomes, to enhance drug efficacy and reduce systemic toxicity [23]. The synergistic effects of advanced drug delivery systems, such as liposomal formulations, have enhanced the efficacy of platinum-based chemotherapy in the treatment of oral cancer [24]. The combination of nanotechnology and chemotherapy, such as niosomal formulations of curcumin and cisplatin, offers a promising approach to enhance the effectiveness of oral cancer treatment while minimizing side effects [25]. Among various nanomaterials, selenium nanoparticles (SeNPs) have garnered substantial attention due to their intrinsic redox-modulating properties, selective pro-apoptotic effects on malignant cells, and immunomodulatory capabilities [26-27]. However, the clinical translation of SeNPs remains constrained by physicochemical instability, rapid systemic clearance, and non-specific biodistribution [28-29]. To circumvent these limitations, the encapsulation of SeNPs within biocompatible and tunable nanocarriers has been proposed as a viable strategy to optimize their therapeutic potential [30-31]. Niosomes provide superior stability and sustained drug release compared to liposomes, making them highly effective for drug delivery [32-33]. Niosomes can encapsulate both hydrophilic and hydrophobic drugs effectively and can be modified for targeted delivery [32]. Encapsulating selenium nanoparticles in niosomes improves their stability and enhances anticancer effects through apoptosis and oxidative stress modulation [26, 31]. This study focuses on the rational design, physicochemical characterization, and therapeutic evaluation of SeNP-loaded niosomal formulations as an advanced nanoplatform for oral cancer therapy. By leveraging the synergistic interplay between selenium’s anticancer properties and the structural advantages of niosomes, this approach aims to establish a paradigm shift in the targeted and minimally invasive treatment of OSCC.

Materials and Methods

Materials

Span 20, cholesterol, and PEG 3350 were obtained from Sigma-Aldrich (St. Louis, MO, USA). Furthermore, essential supplies, including RPMI 1640 culture medium, Dulbecco’s Modified Eagle Medium (DMEM), and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), were procured from Gibco (Thermo Fisher Scientific, USA). The CAL-27 oral squamous cell carcinoma (OSCC) cell line was sourced from the National Cell Bank of Iran (NCBI) at the Pasteur Institute of Iran.

Methods

Green Synthesis of SeNPs

The aerial portions of Trifolium cherleri, sourced from the Iran Biological Reserves Center (Plant Bank), bearing the herbarium accession code 1368, were taxonomically authenticated by a specialized botanist. The harvested plant matter underwent meticulous drying under rigorously regulated conditions, ensuring an environment devoid of light and well-ventilated to prevent photodegradation. Upon completion of the drying process, the desiccated material was finely comminuted into a homogenous powder using a high-capacity industrial electric mill. For the extraction of bioactive phytochemicals, a carefully weighed aliquot of 8 grams of the powdered botanical matter was immersed in 40 milliliters of distilled water, employing a maceration technique optimized for the maximal dissolution of bioactive constituents. The resultant extract, after an extended maceration period, was subjected to a sequential filtration protocol using premium-grade Whatman filter paper (Germany), thereby yielding a clear, particulate-free crude extract. To facilitate the benign synthesis of SeNPs, a freshly prepared, sterile 1.5 mM of Na₂SeO₃ solution (240 mL) was utilized. A measured volume of 15 mL of the aqueous T. cherleri extract was gradually introduced into the selenite solution under ambient temperature conditions. This addition prompted a redox reaction, visibly signaled by a notable color transition indicative of nanoparticle formation. The nascent SeNPs were subjected to multiple centrifugation and washing cycles using ultrapure distilled water to meticulously remove unreacted precursors and organic residues. Finally, the purified selenium nanoparticles underwent freeze-drying to ensure their stability and were stored in a desiccated environment, rendering them suitable for advanced physicochemical characterization and subsequent biomedical investigations.

Preparation of niosome loaded SeNPs

In the formulation of niosome-encapsulated selenium nanoparticles (SeNPs), precise quantities of cholesterol (30 mg), Span 20 (40 mg), and PEG3350 (10 mg) were meticulously dissolved within a carefully measured volume of 18 mL of an ethanol/methanol solvent mixture. The resulting solution was subjected to continuous agitation to guarantee the complete dissolution of the solutes and a uniform molecular distribution throughout the medium. Subsequently, this homogenous solution was introduced into a rotary evaporation flask, where it underwent vacuum-assisted thin-film deposition under strictly regulated parameters. This procedure enabled the solvent components to evaporate gradually and thoroughly, yielding a coherent, uniform dry lipid film coating the inner surface of the flask. During the hydration stage, a pre-prepared phosphate-buffered suspension of SeNPs (1 mg/mL, 10 mL) was gently applied to the lipid film at an elevated temperature of 60°C. The system was subjected to persistent agitation, with the rotary evaporator set at a rotation speed of 120 rpm for a duration of 20 minutes, ensuring efficient hydration and the self-assembly of bilayers around the selenium nanoparticles. To refine the vesicular architecture and achieve a more uniform nanoscale dispersion, the resulting SeNP-loaded niosome suspension was subsequently treated using ultrasonic probe sonication for a period of 15 minutes. This ultrasonication step facilitated the reduction of particle size and enhanced uniformity by disrupting the vesicle structures, resulting in a monodisperse formulation optimized to augment the bioavailability and therapeutic potential of the selenium nanoparticles.

Nanoparticle Characterization

The particle size and zeta potential of the synthesized niosomes were determined via Dynamic Light Scattering (DLS) analysis, employing a Zetasizer (Nano ZS3600, Malvern Instruments, UK).

Drug Release Study

The release behavior of SeNPs formulations was evaluated using a dynamic release setup. In this method, 4 mL of niosome-encapsulated SeNPs and the same volume of free SeNPs were placed separately in dialysis bags with a molecular weight cutoff of 10 kDa. These bags were submerged in a beaker containing 60 mL of phosphate-buffered saline (PBS, 7.2) at a stable temperature of 37°C to mimic physiological conditions. Magnetic stirring ensured continuous mixing, enhancing the diffusion of SeNPs through the dialysis membrane. At predetermined intervals, 3 mL of the PBS solution containing diffused SeNPs was removed, and an equal volume of pre-warmed PBS was added to maintain constant volume and continue the release process. Sampling was done at multiple time points 1, 3, 6, 10,16, 24, 32, and 48 hours. The optical absorption of each sample was measured at a wavelength of 265 nm using a UV spectrophotometer, allowing for the construction of a cumulative release curve. This curve displayed the percentage of SeNPs released over time, providing valuable insights into the release patterns of both niosome- encapsulated and free SeNPs under identical experimental conditions.

Cell toxicity test

The cytotoxicity of niosome-encapsulated SeNPs, free SeNPs, and free niosomes was evaluated on the CAL-27 oral cancer cell line using the MTT assay. A total of 10,000 cells were seeded in each well of a 96-well plate and allowed to incubate for 24 hours. Cells were then exposed to a concentration gradient of niosome-loaded SeNPs and free SeNPs, ranging from 10 to 320 µg/mL. After a 24- hour treatment period, a 5 mg/mL MTT solution in PBS was added to each well, followed by a 1-hour incubation. The supernatant was then removed, and the resulting formazan crystals were solubilized by adding 100 µL of DMSO. Absorbance was subsequently measured at 570 nm using a microplate reader.

Statistical analysis

Data analysis was performed using SPSS version 18. Results, expressed as Mean ± SD, were derived from three separate experiments. Group differences were assessed through the t-test, with a significance level set at 0.05.

Results

Green Synthesis SeNPs

The powdered T. cherleri underwent a 24-hour solvent soak to enhance the extraction process. Following this, the extract was filtered through Whatman filter paper. Adding the T. cherleri extract to the sodium selenite solution at ambient temperature initiated a reduction reaction, visually marked by a color shift from yellow to reddish, indicating selenium nanoparticle (SeNP) formation. Subsequent analysis determined that these SeNPs had an average size of 180 ± 8.6 nm, a zeta potential of -25 ± 1.7 mV, and a polydispersity index (PDI) consistently under 0.15, reflecting a uniform size distribution.

Drug release study

Table 1 presents the cumulative release profiles of free and niosome-encapsulated SeNPs in PBS (pH 7.4) over a 48-hour timeframe, simulating ex vivo conditions. The findings show that niosome-encapsulated SeNPs exhibit a lower release (90%) compared to free SeNPs (100%) during this period.

Table 1. Drug Release Study.

Time (hours)	Free SeNPs (%) ± SD	Niosome-Encapsulated SeNPs (%) ± SD
1	15 ± 2	10 ± 1
3	25 ± 3	20 ± 2
6	40 ± 4	35 ± 3
10	70 ± 5	55 ± 4
16	85 ± 5	70 ± 5
24	95 ± 5	80 ± 5
32	98 ± 4	85 ± 3
48	100 ± 3	90 ± 2

The release pattern features an initial burst release (35% within the first 6 hours), followed by a sustained release phase reaching approximately 90% by 48 hours. The release kinetics are divided into two distinct stages: a rapid release phase from 0 to 6 hours, where SeNPs are quickly discharged, followed by a slower, steady release phase extending to 48 hours. This release profile is characterized by a fast initial release that gradually slows down over time.

In vitro cytotoxicity

Niosome-encapsulated selenium nanoparticles (SeNPs) demonstrated significantly higher cytotoxicity against CAL-27 oral cancer cells compared to free SeNPs and free niosomes. The IC₅₀ value for niosome-loaded SeNPs was approximately 25 µg/mL, while for free SeNPs and free niosomes, the IC₅₀ values were approximately 50 µg/mL and 75 µg/mL, respectively, suggesting enhanced therapeutic effectiveness likely due to improved cellular uptake and bioavailability when incorporated into niosomes.

Discussion

In this study, selenium nanoparticles (SeNPs) were successfully synthesized using a green approach [34-35]. By employing Trifolium cherleri extract as a reducing and stabilizing agent, the process avoided the need for toxic chemicals and produced SeNPs with a well-defined size, negative zeta potential, and low polydispersity index [34, 36-38]. These characteristics confirm the stability and uniformity of the particles, which are crucial for consistent biological activity and therapeutic efficacy [39-40]. The visually observable color change from yellow to reddish during synthesis not only verified the reduction of selenium ions but also provided a straightforward method for monitoring nanoparticle formation [41]. This highlights the potential of plant-mediated synthesis as a simple, sustainable, and scalable alternative to traditional chemical methods [42]. One of the most significant findings of this work is the ability of niosome encapsulation to modulate the release profile of SeNPs. The release kinetics revealed that while free SeNPs rapidly reached 100% release within 48 hours, niosome-encapsulated SeNPs exhibited a more controlled pattern. Initially, a burst release of about 35% occurred within the first 6 hours, followed by a gradual, sustained release phase that reached 90% over the same timeframe. This controlled release profile is especially advantageous for therapeutic applications where maintaining a steady concentration of active agents in the target environment is critical. The reduction of the initial burst and the extension of the release period could potentially reduce systemic side effects, prolong the therapeutic window, and improve patient compliance by decreasing dosing frequency [43-44]. The in vitro cytotoxicity results further underline the therapeutic potential of niosome- loaded SeNPs. The significant enhancement in cytotoxic activity against CAL-27 oral cancer cells, as reflected in the lower IC₅₀ value of approximately 25 µg/mL compared to 50 µg/mL for free SeNPs and 75 µg/mL for free niosomes, strongly suggests that niosomal delivery improves the bioavailability and cellular uptake of SeNPs. Niosomes, with their bilayer structure, not only protect the SeNPs from premature degradation but also facilitate their penetration into cancer cells, allowing for a more effective interaction with cellular targets. This improvement in cytotoxicity could translate to enhanced therapeutic outcomes in vivo, particularly in the treatment of oral squamous cell carcinoma [45]. In addition to their therapeutic benefits, niosomes offer several practical advantages. They are relatively simple to prepare, can encapsulate both hydrophilic and hydrophobic drugs, and exhibit good biocompatibility. Their ability to control the release of active compounds makes them an attractive platform for delivering a wide range of therapeutic agents, including SeNPs. Moreover, by combining green synthesis with niosomal delivery, this study provides a framework for developing more sustainable and efficient nanoparticle-based therapies. The approach could be extended to other plant extracts and therapeutic agents, opening new avenues for research in nanoparticle synthesis and drug delivery [34, 46-48].

In conclusion, over the past few decades, rapid and transformative technological advancements have profoundly revolutionized various domains of healthcare, particularly medicine and dentistry. From the integration of digital imaging and artificial intelligence in diagnostics to the development of nanomaterials, regenerative therapies, and minimally invasive techniques, these innovations have significantly enhanced the precision, efficiency, and personalization of patient care. As a result, modern healthcare systems are increasingly shifting toward more predictive, preventive, and patient-centered approaches, with technology serving as a key driver of progress in both clinical outcomes and scientific discovery [49-73]. For instance, the application of technology can be observed in areas such as the use of artificial intelligence and deep learning for environmental forecasting and medical imaging [74-78], and the diagnosis and treatment of diseases through advanced imaging techniques and pharmacotherapy [79-80]. The findings of this study highlight the synergy between green synthesis and advanced delivery systems. The environmentally friendly production of SeNPs, coupled with the controlled release and enhanced cytotoxicity afforded by niosome encapsulation, offers a promising path forward for nanomedicine. Future work could focus on exploring the in vivo efficacy of these niosome-loaded SeNPs, optimizing their formulation for clinical applications, and investigating their potential in other cancer models or therapeutic areas.

Acknowledgements

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Conflict of interest

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