Green synthesis of AgNPs offers several advantages over traditional physical and chemical methods. Conventional methods often involve high energy inputs, hazardous chemicals, and complex procedures (Subhalakshmi et al., 2024). For example, chemical reduction methods use toxic reducing agents like sodium borohydride, while physical methods such as laser ablation require sophisticated equipment and high energy (Sivalingam and Pandian, 2024). In contrast to conventional synthesis techniques, plant-mediated green synthesis offers an ecologically sustainable, economically viable, and operationally simple approach. By replacing synthetic reagents with phytochemicals, this method circumvents the use of hazardous chemicals and minimizes ecological footprints (Sivalingam et al., 2024c). Furthermore, biogenic AgNPs exhibit superior biocompatibility, as natural capping agents derived from plant extracts reduce cytotoxicity and enhance biosafety in biological systems (Baskaran et al., 2024). Despite these benefits, traditional synthesis routes often achieve finer control over nanoparticle morphology (e.g., size uniformity, geometric precision), prompting ongoing efforts to refine green synthesis protocols for enhanced reproducibility and structural predictability (Pushpanathan et al., 2025).

Plant-based green synthesis represents a transformative paradigm for producing AgNPs with applications in oncology, particularly oral cancer treatment. By elucidating the interplay between phytochemical composition, reaction kinetics, and nanoparticle properties, researchers can engineer AgNPs with customizable physicochemical profiles (Arumugam et al., 2024). Advances in process optimization—such as modulating reaction pH, temperature, and precursor ratios—will enable the scalable production of nanoparticles tailored for targeted drug delivery, photothermal therapy, or diagnostic imaging (Sudhashini et al., 2025). This sustainable methodology not only aligns with global eco-conscious initiatives but also bridges the gap between nanomedicine innovation and clinical translation, offering a roadmap for next-generation cancer therapeutics (Anandapillai et al., 2024).

Optimal plant species for AgNP synthesis are chosen based on their phytochemical richness, geographic availability, and compatibility with biomedical goals (Table 1) (Habeeb Rahuman et al., 2022). Species like Azadirachta indica (neem), Camellia sinensis (green tea), and Ocimum sanctum (tulsi) are prioritized for their high concentrations of antioxidants (e.g., catechins, terpenoids), which accelerate silver ion reduction while imparting therapeutic properties to the nanoparticles (Pugazhendhi et al., 2018; Zhang et al., 2019; Gokulakrishnan et al., 2024; Karunakar et al., 2024; Pauline et al., 2025). Biocompatibility assessments further guide selection, ensuring minimal cytotoxicity toward healthy tissues and alignment with drug delivery requirements. This strategic selection enhances nanoparticle functionality for applications such as targeted cancer therapy (David et al., 2021; Agarwal et al., 2025).

Optimization of synthesis conditions requires quantitative control of precursor concentration, extract ratios, pH, temperature, and reaction duration (Table 2) (Sivalingam et al., 2024b). For example, most reproducible studies utilize AgNO₃ concentrations between 0.five to five mM, extract-to-metal salt ratios ranging from 1:5 to 1:20, and alkaline pH (9–11), which enhances polyphenol-mediated reduction (Habib et al., 2024). Temperature ranges of 40 °C–80 °C significantly accelerate reaction kinetics and improve nucleation uniformity. These quantitative boundaries are essential for achieving consistent AgNP morphology and have been incorporated into this revised review to enhance methodological rigor and reproducibility (Balamurugan et al., 2024; Bekele et al., 2024; Hemavathy et al., 2024; Nandhini et al., 2024; Sangameshwaran et al., 2024).

The plant-mediated green synthesis of AgNPs leverages the redox activity of phytochemicals to convert ionic silver (Ag⁺) into metallic nanoparticles (Ag⁺ → Ag⁰). This process initiates with the preparation of aqueous or solvent-based plant extracts, where heating plant materials (e.g., leaves, roots, or flowers) releases bioactive constituents such as polyphenols, flavonoids, and proteins (Figure 1) (Brindhadevi et al., 2021; Kaliaperumal et al., 2023; Muruganandham et al., 2023). These biomolecules act as dual-function agents: reducing silver ions to nucleate nanoparticles and coating their surfaces to regulate growth and prevent aggregation (Subramaniam et al., 2021; Kingslin et al., 2023). The phytochemical diversity across plant species leads to variations in nanoparticle morphology (e.g., spherical, triangular) and colloidal stability, underscoring the role of specific metabolites in directing synthesis kinetics (Dilipan et al., 2023; Lavanya et al., 2023; Vijayaraj et al., 2023).

In vitro analyses are pivotal for assessing the antineoplastic potential of plant-derived AgNPs against oral carcinoma (Table 3) (Budi and Farhood, 2023). These investigations focus on cellular responses—such as viability, apoptotic induction, ROS generation, and cell cycle arrest—in oral cancer lines (e.g., SCC-4, SCC-9, CAL 27, KB) following exposure to varying AgNP concentrations. Such studies elucidate mechanisms of action and therapeutic windows, guiding translational development (Subramanyam et al., 2021; Shukla and Senapathya, 2025).

Subramanyam et al. (2023) reported the green synthesis of AgNPs using Byttneria herbacea leaf extract (BH-AgNPs), emphasizing their application in oral oncology. UV-Vis spectroscopy revealed a time-dependent surface plasmon resonance (SPR) redshift from 422 nm to 437 nm, confirming nanoparticle maturation. TEM and DLS analyses identified spherical BH-AgNPs with an average diameter of 8 nm and high colloidal stability (zeta potential: 21 mV). The nanoparticles exhibited robust antioxidant activity, neutralizing free radicals, and potent antibacterial effects against Escherichia coli and Staphylococcus aureus. In KB oral cancer cells, BH-AgNPs demonstrated significant cytotoxicity (IC₅₀: 89.25 μL/mL), inducing apoptosis via ROS-mediated pathways. These findings position BH-AgNPs as a dual-functional platform for oral cancer therapy, merging anticancer efficacy with antimicrobial protection (Subramanyam et al., 2023).

Barua et al. (2017) synthesized AgNPs using Thuja occidentalis leaf extract, highlighting their biocompatibility and anticancer versatility. The spherical nanoparticles (12.7 nm average size) exerted concentration-dependent cytotoxicity (6.25–50 μg/mL) against breast (MCF-7, MDA-MB-231), cervical (HeLa), and oral (KB) cancer lines, while sparing normal human PBMCs and rat hepatocytes. This selectivity underscores their biosafety profile. Additionally, the AgNPs displayed broad-spectrum antibacterial activity against pathogens like Staphylococcus aureus and Pseudomonas aeruginosa. The study underscores the potential of phytogenic AgNPs as multitarget agents, capable of synergizing anticancer and antimicrobial actions with minimal off-target toxicity (Barua et al., 2017). Both studies underscore the therapeutic potential of plant-synthesized AgNPs in oral cancer, highlighting key advantages including eco-sustainable synthesis via plant extracts that reduce reliance on toxic reagents, multifunctionality through dual anticancer and antimicrobial actions to address comorbidities like infections, and enhanced biocompatibility due to natural capping agents that minimize off-target cytotoxicity, thereby improving therapeutic indices. These preclinical findings emphasize the need for further in vivo validation and clinical trials to translate phytogenic AgNPs into next-generation, precision-based therapies for oral oncology, bridging green chemistry innovations with unmet clinical needs.

Fundamental evaluations of biogenic AgNPs in oral cancer research prioritize cell viability assays, such as MTT, MTS, and Alamar Blue, which quantify metabolic activity through enzymatic reduction of tetrazolium salts into formazan. These assays reveal concentration-dependent reductions in viability, reflecting AgNP-induced cytotoxicity, where higher nanoparticle concentrations correlate with diminished cancer cell survival (Karthikeyan et al., 2024; Rathi and Ramesh, 2024; Vanti et al., 2024).

Recent studies highlight the therapeutic versatility of plant-synthesized AgNPs. Alsareii et al. (2022) utilized Rhizophora apiculata leaf extract to produce AgNPs (35–100 nm) via eco-friendly methods, demonstrating potent antioxidant, anti-inflammatory, and cytotoxic effects against oral, lung, and skin cancer lines. Enhanced wound closure and cell migration further underscored their biomedical potential (Alsareii et al., 2022). Similarly, Huang Fang (2022) synthesized AgNPs (26.11 nm average size) from Rheum ribes L. leaves, observing significant dose-dependent cytotoxicity in oral cancer cells (HSC-2, HSC-3, HSC-4) at IC₅₀ values of 125–250 μg/mL, linked to their antioxidant activity (Fang, 2022). Yan et al. (2022) reported spherical AgNPs (50–90 nm) from Salvia officinalis, which selectively targeted oral squamous carcinoma cells (KB, HSC-2, HSC-3, Ca9-22) while sparing normal HUVEC cells, with cytotoxicity attributed to ROS modulation and antioxidant mechanisms (Yan et al., 2022). Collectively, these studies validate the role of green synthesis in producing biocompatible, multifunctional AgNPs. Characterization via SEM, XRD, and FTIR confirmed structural integrity and phytochemical capping, while dose-responsive cytotoxicity and selective action against malignant cells highlight their therapeutic promise. The integration of antioxidant properties with anticancer efficacy positions plant-derived AgNPs as innovative candidates for oral oncology, warranting clinical exploration to translate preclinical findings into targeted therapies.

Beyond assessing cell viability, researchers analyze morphological transformations in cancer cells to evaluate nanoparticle-mediated toxicity. Microscopy-based assessments reveal structural anomalies such as cytoplasmic condensation, membrane protrusions (blebbing), and chromatin fragmentation—hallmark indicators of apoptotic pathways—or cellular swelling and membrane rupture suggestive of necrotic processes. These visual biomarkers corroborate the cytotoxic impact of AgNPs, offering spatial insights into cell death mechanisms (Oviya et al., 2023; Paramasivam et al., 2023).

To differentiate apoptotic from necrotic outcomes, advanced assays are employed. Flow cytometry using Annexin V/propidium iodide dual staining quantifies phosphatidylserine externalization (apoptosis) versus membrane permeability (necrosis). Complementary techniques like TUNEL assays detect DNA strand breaks, while caspase activation profiling (e.g., caspase-3/7 activity) maps enzymatic pathways driving programmed cell death. These methods collectively validate apoptosis as a primary mode of AgNP-induced cytotoxicity (Sabirova et al., 2025).

ROS generation is a critical mediator of AgNP toxicity. Elevated intracellular ROS flux, measured via fluorogenic probes (e.g., DCFH-DA), disrupts redox equilibrium, inducing oxidative damage to lipids, proteins, and DNA. This oxidative stress activates stress-responsive kinases (e.g., JNK, p38 MAPK) and mitochondrial permeability transition, culminating in caspase-dependent apoptosis (Ashique et al., 2022; Harsha et al., 2022; Loganathan et al., 2022).

Cell cycle modulation further elucidates AgNP anticancer mechanisms. Flow cytometric DNA content analysis identifies phase-specific arrests (e.g., G0/G1, S, or G2/M), linking nanoparticle exposure to proliferative inhibition. Such arrests often correlate with cyclin-dependent kinase suppression or checkpoint kinase activation, providing mechanistic targets for therapeutic optimization (Garapati et al., 2022).

Dziedzic et al. investigated nanoscale AgNPs (10 nm) against SCC-25 tongue carcinoma, revealing dose- and time-dependent antiproliferative effects (IC₅₀: 5.19 μg/mL at 48 h). AgNPs induced G0/G1 phase arrest and upregulated pro-apoptotic Bax/Bcl-2 ratios, triggering mitochondrial apoptosis. Intriguingly, co-treatment with berberine—an isoquinoline alkaloid—attenuated AgNP cytotoxicity at higher doses, suggesting antagonistic interactions potentially mediated by nanoparticle-alkaloid complexation. This finding underscores the need to evaluate combinatorial regimens, as phytochemical adjuvants may inadvertently diminish AgNP efficacy (Dziedzic et al., 2016). These methodologies—morphological analysis, apoptosis profiling, ROS quantification, and cell cycle tracking—collectively decode AgNP bioactivity. While AgNPs alone exhibit potent, ROS-driven anticancer effects, their interplay with co-administered compounds like berberine necessitates careful evaluation to optimize therapeutic outcomes in oral oncology.

The anticancer effects of green-synthesized silver nanoparticles (AgNPs) against oral cancer cells are controlled not by general mechanisms like ROS generation or mitochondrial depolarization alone, but by highly specific physicochemical properties of the nanoparticles themselves. Key factors such as particle size, shape, surface charge, ligand chemistry, dissolution kinetics, and the phytochemical corona collectively determine how AgNPs interact with cancer cell membranes, organelles, and intracellular signaling pathways. For example, smaller AgNPs (<20 nm) display heightened cellular uptake and ROS production, enabling deeper penetration into cells and targeting mitochondria and the nucleus, while larger particles (>40–50 nm) accumulate in lysosomes, causing lysosomal stress rather than immediate mitochondrial apoptosis (Ejaz et al., 2023). The morphology of AgNPs influences uptake and cellular stress, as spherical forms typically internalize more efficiently than rods or triangles, which can disrupt cell membranes and cytoskeleton. Surface charge, mainly set by the phytochemical corona, guides selective binding to more positively charged cancer cell membranes, facilitating preferential uptake and cytotoxicity; negatively charged AgNPs target malignant cells, while rare positively charged ones, usually protein-capped, can harm both healthy and cancerous cells indiscriminately. The corona, rich in plant-derived compounds, shapes redox properties, ion release, and intracellular fate—phenolic-rich coronas increase ROS and Ag⁺ ion release, amplifying DNA and protein damage, while protein- or polysaccharide-rich coronas slow dissolution and promote distinct death pathways. Ligand chemistry further directs nanoparticle interaction with mitochondrial proteins or DNA, fine-tuning the type and extent of damage. Ultimately, these attribute-dependent mechanisms account for nuanced variations in cytotoxicity, mitochondrial disruption, oxidative bursts, and suppression of DNA repair in oral cancer cells, making the biological impacts of AgNPs emergent behaviors dictated by their unique physicochemical identities rather than universal modes of action (Balu et al., 2022; David et al., 2022; Padmanabhan et al., 2022).

Inbakandan et al. (2016) pioneered the ultrasonic-assisted biosynthesis of AgNPs using Haliclona exigua marine sponge extract, yielding unique flower-like nanocolloids (100–120 nm). These nanoparticles demonstrated potent antibacterial action against oral pathogens (S. aureus, Streptococcus mitis) and selective cytotoxicity toward KB oral cancer cells (IC₅₀: 0.6 μg/mL), outperforming chemically synthesized counterparts. The enhanced bioactivity was attributed to phytochemical capping (terpenoids, alkaloids) and nanoscale surface interactions, which amplified ROS generation and membrane permeabilization in malignant cells (Inbakandan et al., 2016).

Ganesan A. et al. (2024) harnessed Solanum trilobatum leaf extract to synthesize spherical AgNPs (20 nm) stabilized by polyphenolic ligands. These nanoparticles suppressed OSCC proliferation by downregulating PI3K/Akt/mTOR signaling, evidenced by reduced phosphorylated Akt levels and mitochondrial depolarization. DNA laddering assays confirmed apoptosis induction, while negligible cytotoxicity in non-malignant cells highlighted their therapeutic window (Figure 2). The study positions plant-capped AgNPs as dual-targeting agents capable of bypassing chemoresistance mechanisms in oral oncology (Ganesan A. et al., 2024). AgNPs co-opt both mitochondrial (intrinsic) and death receptor (extrinsic) apoptosis pathways. Intrinsic activation involves cytochrome c-mediated caspase-9 signaling, while extrinsic triggers engage cell surface receptors (e.g., Fas, TNF-R1), activating caspase-8. Cross-talk between these pathways via Bid protein cleavage ensures robust apoptosis execution, circumventing cancer cell evasion tactics. Such dual-pathway engagement underscores AgNPs’ versatility in overcoming therapeutic resistance (Loppnow et al., 2013).

The evaluation of biogenic AgNPs against conventional chemotherapy highlights their potential as alternative or adjunct therapies for oral cancer. Traditional chemotherapeutic agents, though effective in targeting malignant cells, are frequently associated with adverse effects and the emergence of resistance. In contrast, biogenic AgNPs present advantages in efficacy, specificity, and safety, positioning them as a promising therapeutic innovation (Zhou et al., 2025).

Research indicates that AgNPs exhibit cytotoxic effects on oral cancer cells equal to or exceeding those of standard chemotherapeutics. Notably, AgNPs induce apoptosis and reduce viability in cell lines such as SCC-4, SCC-9, CAL 27, and KB, often at lower concentrations than conventional drugs. This heightened potency could minimize dosage requirements and mitigate side effects in clinical applications (Subramanyam et al., 2021; Shukla and Senapathya, 2025).

A 2022 investigation by Halkai et al. examined AgNPs synthesized using Fusarium semitectum fungi, assessing their impact on oral squamous cell carcinoma (SCC-9) via MTT assays. The study revealed concentration-dependent suppression of SCC-9 proliferation, with an IC50 of 12 μL/mL. At 50 μL/mL, cell growth inhibition reached 88.46%, underscoring robust antitumor activity. These findings align with prior work emphasizing the enhanced biological performance of biosynthesized AgNPs, attributed to targeted mechanisms such as oxidative stress induction and cellular disruption. The study reinforces the viability of fungal-derived AgNPs as therapeutic candidates, advocating for eco-friendly synthesis methods in oncology (Halkai et al., 2022).

Similarly, Ghabban et al. (2022) developed AgNPs using Astragalus spinosus extract, evaluating their antibacterial and cytotoxic properties. The nanoparticles demonstrated strong antimicrobial activity against Streptococcus mutans and Actinomyces viscosus, with MIC and MBC values between 10.6 and 26.6 μg/mL. Mechanistic studies linked this activity to ROS generation and subsequent release of cellular components like nucleic acids. Cytotoxicity assays further revealed selective toxicity toward SCC4 oral cancer cells over NOF18 normal cells, highlighting their potential for targeted therapy. This dual functionality positions plant-derived AgNPs as versatile tools in combating oral pathologies while minimizing harm to healthy tissues (Ghabban et al., 2022).

A key benefit of biogenic AgNPs lies in their tumor-selective targeting, facilitated by the enhanced permeability and retention (EPR) effect. Tumor vasculature’s irregular architecture and compromised lymphatic drainage promote nanoparticle accumulation, while cancer cells’ distinct membrane properties enhance AgNP uptake. This specificity reduces off-target toxicity, addressing a major limitation of conventional therapies that indiscriminately affect healthy tissues (Kovács et al., 2022). Biogenic AgNPs also exhibit a favorable safety profile compared to agents like cisplatin and 5-fluorouracil, which are linked to kidney toxicity, nerve damage, and bone marrow suppression. Preclinical studies suggest plant-synthesized AgNPs display lower toxicity toward non-malignant cells, potentially improving patient tolerance and quality of life during treatment (Nikolova et al., 2023). Furthermore, AgNPs may circumvent chemoresistance mechanisms, such as drug efflux or DNA repair upregulation, through multifaceted actions including ROS generation, mitochondrial dysfunction, and signaling pathway interference. These diverse mechanisms reduce the likelihood of resistance, enhancing therapeutic durability (Do et al., 2025). Biogenic AgNPs represent a viable option for oral cancer therapy, combining potent anticancer activity with selective targeting and reduced toxicity. While preclinical data are encouraging, translational success requires further validation through in vivo studies and clinical trials to establish safety, efficacy, and dosing protocols.

In vivo validation is crucial for translating the promising in vitro anticancer effects of biogenic AgNPs into clinical practice. Various animal models have been employed to evaluate their therapeutic efficacy, biodistribution, and safety in oral cancer treatment.

Xenograft Models: Human oral squamous cell carcinoma (OSCC) cells (e.g., SCC-9, CAL 27) implanted subcutaneously or orthotopically into immunocompromised mice serve as prevalent preclinical models (Ren et al., 2024). For instance, Subramanyam et al. (2023) reported that intravenous administration of Byttneria herbacea-derived AgNPs at doses of 10 mg/kg body weight thrice weekly for 21 days significantly reduced tumor volume by approximately 62% compared to untreated controls, without observable systemic toxicity. Histopathological analyses revealed extensive tumor necrosis and apoptosis, confirming antitumor activity (Subramanyam et al., 2023). Similarly, Jadhav et al. (2018) demonstrated that AgNPs biosynthesized using Salacia chinensis bark extract, administered intraperitoneally at 5 mg/kg daily for 2 weeks in SCC xenograft-bearing mice, inhibited tumor growth by 54% and decreased proliferation marker Ki-67 expression, while sparing normal tissue architecture (Jadhav et al., 2018).

Syngeneic and Orthotopic Models: Orthotopic implantation of mouse oral cancer cells into immunocompetent mice offers tumor microenvironment fidelity and immune system engagement (Tinajero-Diaz et al., 2021; Zhou et al., 2025). In one study, AgNPs synthesized from Solanum trilobatum leaf extract were administered intratumorally at 3 mg/kg weekly over 4 weeks, resulting in a 70% tumor volume reduction and enhanced survival rates compared to controls. Immune profiling indicated increased infiltration of cytotoxic T cells, suggesting immunomodulatory benefits (Ganesan A. et al., 2024).

Genetically Engineered Mouse Models (GEMMs): Although less commonly applied to AgNP studies, GEMMs recapitulate spontaneous tumorigenesis. Preliminary findings indicate that chronic oral administration of AgNPs at 2 mg/kg for 8 weeks delayed tumor onset and progression, supporting their chemopreventive potential (Janzadeh et al., 2022).

Understanding the in vivo fate of green-synthesized silver nanoparticles requires more than descriptive summaries of animal experiments; it necessitates an analysis of how nanoparticle size, surface chemistry, corona composition, and dose determine biodistribution, accumulation, clearance, and systemic toxicity. Biodistribution studies using ICP-MS consistently report that biogenic AgNPs exhibit preferential accumulation in the liver and spleen, with hepatic concentrations reaching 3%–8% of the administered dose per Gram of tissue within 24 h. This reflects the dominant role of Kupffer cells in nanoparticle sequestration. Smaller nanoparticles (<20 nm) typically show higher renal accumulation due to enhanced glomerular filtration, while larger AgNPs (>40 nm) accumulate primarily in the liver and spleen due to macrophage-driven uptake. (Haripriyaa and Suthindhiran, 2023).

Tumor accumulation is driven by the enhanced permeability and retention (EPR) effect, with biogenic AgNPs exhibiting 5–10-fold higher tumor uptake relative to chemically capped counterparts, likely due to the presence of hydrophilic phytochemical coronas. Comparative biodistribution analyses show that nanoparticles capped with polyphenolic coronas accumulate more efficiently in tumors but also display altered hepatic retention dynamics due to increased protein binding in circulation (Cai et al., 2023).

Clearance mechanisms of biogenic AgNPs depend strongly on particle size and corona chemistry. Particles smaller than 10 nm are predominantly cleared through renal excretion, with up to 40%–50% of the injected dose detected in urine within 72 h. By contrast, larger nanoparticles undergo hepatobiliary clearance, with fecal elimination representing a major excretion route. Corona composition also modulates clearance: protein-rich coronas slow systemic elimination due to increased opsonization, whereas polyphenol-dense coronas facilitate more rapid clearance and reduced long-term tissue retention (Jain and Bhise, 2025).

Organ-specific toxicity profiles reflect these biodistribution patterns. Hepatic toxicity is most frequently reported, typically emerging at doses above 10–20 mg/kg in mice, and is characterized by elevated ALT/AST levels, mild steatosis, and centrilobular inflammation. Renal toxicity becomes relevant for smaller AgNPs due to their efficient renal filtration, with dose-dependent tubular degeneration reported at higher exposure levels. Splenic accumulation may lead to marginal zone expansion or increased oxidative stress, although these effects are generally mild at pharmacologically relevant doses. These findings are consistent with broader nanotoxicology literature, including ferric oxide nanoparticle studies, which emphasize macrophage sequestration, oxidative stress markers, and organ-specific histopathology as key endpoints for safety assessment (Anwaar et al., 2024b).

Dose–response trends underscore the dual nature of AgNPs as both therapeutic and potentially toxic agents. At lower doses (one to five mg/kg), most biogenic AgNPs exhibit minimal systemic toxicity and demonstrate effective tumor growth suppression in xenograft models. However, doses above 20–30 mg/kg may induce oxidative stress, mitochondrial dysfunction, and inflammatory responses in non-target organs, particularly in the liver and kidney. These outcomes highlight the necessity of carefully optimizing dose regimens based on nanoparticle size, surface chemistry, and corona stability (Khan et al., 2025).

Overall, the pharmacokinetic and biodistribution behavior of green-synthesized AgNPs is shaped by a complex interplay between physicochemical characteristics and biological systems. A critical understanding of these interactions is essential for evaluating therapeutic potential, anticipating systemic impacts, and establishing safe translational pathways.

Efficacy: Multiple in vivo investigations demonstrate that biogenic AgNPs exert dose-dependent tumor growth suppression, apoptosis induction, and metastasis inhibition in oral cancer models (Noga et al., 2023). For example, Alsareii et al. (2022) observed that oral administration of Rhizophora apiculata leaf extract-mediated AgNPs (10 mg/kg daily for 3 weeks) reduced tumor volume by 65%, associated with diminished expression of proliferation markers (PCNA) and enhanced caspase-3 activity. Combining AgNPs with low-dose cisplatin synergistically enhanced tumor regression while reducing cisplatin-associated toxicity (Alsareii et al., 2022).

Safety: Comprehensive toxicity assessments include histopathology of major organs (liver, kidney, heart, spleen), blood biochemical analyses, and hematological profiling. Across studies, biogenic AgNPs exhibit minimal organ toxicity at therapeutic doses (Barua and Buragohain, 2024; Dias et al., 2025). Subramanyam et al. (2023) reported no significant changes in liver enzymes (ALT, AST) or renal markers (creatinine, BUN) in treated mice. Hemolysis and coagulation assays confirmed hemocompatibility. Immunological analyses showed no acute hypersensitivity or immunosuppression (Kah et al., 2023). Long-term exposure studies extending to 90 days report no mortality or severe adverse events but highlight the necessity to monitor for possible bioaccumulation and subtle organ function alterations in chronic use (Noga et al., 2023).

While promising, challenges remain for clinical translation. Variability in nanoparticle size, shape, and surface chemistry due to green synthesis protocols can affect reproducibility and pharmacodynamics. Scaling up production with stringent quality control is essential (Teh et al., 2024). Animal models, especially immunocompromised xenografts, do not fully replicate human immune-tumor interactions. Thus, synergy effects and immunomodulation require validation in more representative models (Saker et al., 2024). Furthermore, precise dosing regimens, nanoparticle stability in physiological conditions, and potential long-term toxicity—including genotoxicity and environmental impact—necessitate thorough investigation (Kah et al., 2023). Clinical trials must establish standardized protocols for administration routes, dosage, and patient selection based on biomarkers such as tumor receptor expression and HPV status to maximize safety and efficacy (Ahmed et al., 2022; Dias et al., 2025).

Biocompatibility assessment is crucial for determining whether biogenic AgNPs are compatible with biological systems and can be safely used in medical applications. This process involves several key evaluations to ensure that AgNPs do not cause harmful effects to cells, tissues, or the immune system (Venkatesan et al., 2018).

Cell Viability Assays are critical tools for evaluating the effects of AgNPs on cellular health. These assays often employ in vitro models, including fibroblasts, epithelial cells, and endothelial cells, to analyze nanoparticle-induced cytotoxicity. Common methods such as the MTT, MTS, and Alamar Blue assays quantify cellular metabolic activity, offering insights into the dose-dependent toxicity of AgNPs. By identifying concentrations that balance therapeutic effectiveness with minimal harm, these assays guide the establishment of safe nanoparticle dosing guidelines (Abitha et al., 2019).

A 2023 investigation by Pourhaji et al. examined the eco-friendly synthesis of AgNPs using Lactobacillus acidophilus, a probiotic bacterium, and assessed their anticancer potential against oral squamous cell carcinoma (OSCC) cells. This study reflects the expanding role of nanotechnology in medicine, particularly in oncology. The biosynthesized AgNPs were validated through visual color shifts, UV-Vis spectroscopy, and advanced imaging techniques (TEM/SEM), which confirmed their spherical morphology and average size of 397 nm. Using the MTT assay, the researchers observed a concentration-dependent reduction in OSCC cell survival, underscoring the nanoparticles’ therapeutic promise for oral cancer. This work aligns with broader research on green synthesis methods, such as those utilizing plant extracts, which prioritize eco-conscious and biocompatible synthesis techniques. While Pourhaji et al. focused on bacterial synthesis, their findings resonate with plant-based approaches by emphasizing reduced environmental impact and enhanced biocompatibility. Both strategies aim to advance oral cancer treatment by developing safer, more effective AgNP-based therapies (Pourhaji et al., 2023). The integration of such sustainable methods highlights their potential to revolutionize oncology, offering alternatives to conventional, often toxic, cancer treatments.

Hemocompatibility studies are essential for understanding how AgNPs interact with blood components. Blood compatibility assays focus on various aspects, including the potential for hemolysis, which is the lysis of red blood cells (RBCs). Hemolysis assays test whether AgNPs cause RBC rupture, which can lead to adverse reactions in the bloodstream. Additionally, clotting assays are conducted to evaluate the impact of AgNPs on blood coagulation, ensuring that they do not interfere with normal clotting processes and cause unwanted bleeding or clotting disorders (Adhikari et al., 2019; Shravan Kumar and Thangavelu, 2019; Vignesh et al., 2019).

Immunological Compatibility is another critical aspect of biocompatibility assessment. This involves studying the effects of AgNPs on immune cells such as macrophages, dendritic cells, and lymphocytes. Researchers examine cytokine secretion profiles, phagocytosis activities, and immune cell activation to ensure that AgNPs do not elicit adverse immune responses. By evaluating these parameters, it is possible to confirm that AgNPs do not trigger hypersensitivity reactions, excessive inflammation, or other immune system disturbances (Chithralekha and Rajeshkumar, 2019).

Overall, a comprehensive biocompatibility assessment involves these multiple approaches to ensure that AgNPs are safe for use in medical applications. By carefully evaluating cell viability, hemocompatibility, and immunological compatibility, researchers can ensure that AgNPs do not pose significant risks to human health and can be utilized effectively in various therapeutic contexts. This thorough evaluation is critical for the successful translation of AgNPs from preclinical research to clinical use, ensuring they provide beneficial effects without causing harm.

While biogenic AgNPs are recognized for their potent cytotoxic effects against cancer cells, it is crucial to thoroughly evaluate their potential toxicity to normal cells to ensure their safe application in clinical settings. Assessing selective toxicity is a primary focus, aiming to determine how preferentially AgNPs target cancer cells compared to normal cells. This is achieved by comparing cytotoxicity profiles across various cancer and normal cell lines, allowing researchers to identify AgNPs that exhibit favorable selectivity indices. Selective toxicity is desirable as it implies that the nanoparticles can effectively target and kill cancer cells while minimizing harm to healthy cells (Ramzan et al., 2022).

In addition to selective toxicity, researchers must address non-specific toxicity, which involves understanding how AgNPs might impact normal cells through unintended mechanisms. AgNPs promote oxidative stress through the generation of ROS, which disrupt critical cellular structures such as DNA, proteins, and lipids. This oxidative damage can result in apoptosis or functional impairment, even in healthy cells. To evaluate such non-selective toxicity, researchers analyze alterations in cell shape, survival rates, and biological activity following AgNP exposure. These assessments help determine the balance between therapeutic benefits and unintended harm, guiding the development of safer nanoparticle-based treatments. Such evaluations are essential for identifying any adverse effects that might arise from exposure to AgNPs (Gayathri et al., 2019).

Organ-specific toxicity is another critical area of concern. To evaluate how AgNPs may affect specific organs, researchers use organotypic models or ex vivo organ culture systems. These models allow for the study of AgNPs’ impact on vital organs like the liver, kidneys, lungs, and brain, providing insights into potential organ-specific responses. For instance, the liver and kidneys are critical for metabolizing and excreting nanoparticles, making them particularly relevant for toxicity studies. Similarly, assessing the effects on lungs and brain helps determine if AgNPs might pose risks in these sensitive areas (Suresh et al., 2019).

Overall, a comprehensive evaluation of AgNPs’ potential toxicity to normal cells involves understanding both their selective and non-selective effects, as well as their impact on specific organs. This thorough approach is essential for ensuring that AgNPs can be safely used in therapeutic contexts without causing significant harm to normal tissues, thus paving the way for their successful clinical application.

Long-term experimental studies in animal models have provided critical insights into the potential risks associated with chronic exposure to AgNPs, including those of biogenic origin. Rather than relying on speculation, recent evidence delineates the mechanistic pathways through which such exposures could lead to adverse effects involving genotoxic, neurotoxic, and hepatotoxic outcomes (Sati et al., 2025).

Genotoxicity: Multiple in vivo studies confirm that prolonged oral or parenteral exposure to AgNPs induces significant genotoxic effects in mammals. In Sprague-Dawley rats, for example, repeated oral administration of AgNPs at doses ranging from 5 to 100 mg/kg/day over 5 days resulted in substantial DNA damage in bone marrow cells, increased chromosomal aberrations, heightened micronuclei frequency, and reduced mitotic index. Mechanistically, these effects correlate with an upsurge of reactive oxygen species (ROS), initiating oxidative stress and directly damaging genomic DNA. Review analyses consistently demonstrate that AgNPs produce genotoxic effects at all examined endpoints, including in vivo micronucleus formation, chromosome aberration, and comet assays in mammals. The extent of DNA damage is influenced by nanoparticle characteristics (size, coating), but genotoxicity persists across both biogenic and chemically synthesized AgNPs (Patlolla et al., 2015; Rodriguez-Garraus et al., 2020).

Neurotoxicity: Long-term AgNP exposure has also been implicated in neurotoxicity, predominantly following chronic oral or intravenous administration. In a rigorously controlled mouse study, daily oral dosing with polyvinylpyrrolidone (PVP)-coated AgNPs over 30, 60, 120, and 180 days led to a cumulative, region-specific accumulation of silver in brain tissues—most notably in the hippocampus, cortex, and cerebellum (Wang et al., 2023). Histopathological analysis revealed loss and degeneration of hippocampal CA2 neurons after 120–180 days, which was not observed in other brain regions. Functionally, exposed animals developed impaired social interaction, reduced exploratory activity, and memory deficits, correlating with neuronal injury and disruption of memory-associated structures. Mechanistically, AgNPs cross the blood–brain barrier, provoke persistent oxidative stress, disrupt neuronal integrity, and impair synaptic signaling, collectively resulting in behavioral changes and long-term memory impairment (Wang et al., 2023).

Hepatotoxicity: Persistent AgNP exposure can lead to pronounced hepatotoxicity in animal models. In studies with Sprague Dawley rats, daily intraperitoneal injections of AgNPs at 0.25–1 mg/kg over 15 or 30 days produced dose- and time-dependent liver damage. Documented effects included reduced body and organ weights, elevated serum markers of hepatic injury, and characteristic histopathological lesions—such as cholangiopathy, hepatocellular necrosis, sinusoidal congestion, and Ito cell proliferation. Mechanistically, AgNP-induced hepatotoxicity is mediated by two pathways; Oxidative Stress and Apoptosis: Increased ROS and nitrosative stress deplete glutathione (GSH), upregulate pro-apoptotic factors (BAX, caspase-3), and suppress anti-apoptotic markers (Bcl-2), leading to mitochondrial dysfunction and extensive hepatocyte apoptosis. Fibrosis and Inflammation: Chronic exposure enhances expression of fibrogenic (TGF-β1, α-SMA) and pro-inflammatory (iNOS) genes, activating stellate cells and triggering hepatic fibrosis. Repeated findings confirm these mechanisms are consistent regardless of nanoparticle synthesis route, affirming that biogenic AgNPs also warrant careful, long-term toxicological evaluation (Assar et al., 2022).

Scalable and clinically compliant production of green-synthesized silver nanoparticles presents significant technical challenges that extend far beyond the conceptual advantages of sustainability and biocompatibility. The primary barrier lies in the intrinsic phytochemical variability of plant extracts, which is influenced by species, geographic origin, season, soil composition, plant age, extraction solvent, and environmental stress conditions (Kordasht et al., 2025). These variations profoundly affect the concentration of key reducing and stabilizing metabolites, including polyphenols, flavonoids, terpenoids, proteins, and organic acids. As a result, nucleation rates, particle size distribution, surface charge, corona composition, and ultimately the biological activity of AgNPs may differ substantially between batches. Achieving clinical-grade reproducibility therefore requires standardized cultivation or controlled sourcing of plant material, quantitative phytochemical fingerprinting (e.g., HPLC, LC–MS), and the establishment of validated extract specifications before synthesis (Muslim et al., 2025).

Purification represents another major technical bottleneck in GMP manufacturing. Plant-mediated reactions introduce complex organic residues, unbound phytochemicals, polysaccharides, and protein fragments that must be removed to meet regulatory purity standards. Laboratory-scale purification using centrifugation or simple washing steps is insufficient for medical applications. GMP-grade workflows require scalable purification technologies such as tangential flow filtration (TFF), membrane ultrafiltration/diafiltration, chromatographic fractionation, and sterile 0.22 µm filtration to ensure consistent removal of organic contaminants. In addition, plant-derived endotoxins and microbial by-products pose safety risks; thus, validated endotoxin quantification using LAL or recombinant Factor C assays is essential for intravenous or intratumoral applications (Guleria et al., 2022).

Reproducibility across manufacturing batches must be verified through comprehensive analytical characterization. This includes particle size and charge (DLS, TEM, NTA, zeta potential), corona chemical composition (FTIR, LC–MS), residual solvent or impurity analysis, and batch-release specifications similar to those used for biologics and nanomedicines. Studies on biogenic copper oxide nanoparticles have demonstrated that synthesis chemistry and purity directly influence biological activity and toxicity profiles; similar consistency must be demonstrated for biogenic AgNPs before clinical progression (Anwaar et al., 2024a).

Long-term stability and shelf-life are additional critical considerations, as green-synthesized nanoparticles are highly sensitive to oxidation, agglomeration, and corona degradation. The phytochemical corona may undergo desorption, denaturation, or oxidation during storage, altering colloidal stability and bioactivity. Stability-indicating assays under ICH Q1A (R2) guidelines—including accelerated stability testing, freeze–thaw assessment, oxidative stress testing, and long-term storage evaluation—are required to determine appropriate storage conditions and validate shelf-life. Stabilization strategies such as cryoprotectants, antioxidant additives, lyophilization, or PEGylation may be necessary to maintain physicochemical integrity during extended storage (Abegunde et al., 2024).

Finally, GMP translation demands rigorous documentation, validated standard operating procedures, and compliance with regulatory frameworks such as ICH Q8/Q9, FDA nanomaterial guidelines, and EMA reflection papers. These include critical quality attributes (CQAs), critical process parameters (CPPs), continuous process monitoring, and risk-based quality assessment. Without these layers of standardization and documentation, green-synthesized AgNPs cannot meet clinical or commercial manufacturing requirements (Edo et al., 2025).

Incorporating AgNPs into conventional cancer therapies offers opportunities to enhance treatment efficacy but requires careful strategy. Combining AgNPs with chemotherapy, radiotherapy, or immunotherapy could exploit synergistic mechanisms—for example, sensitizing drug-resistant tumors or amplifying immune responses through antimicrobial effects (Kailasa et al., 2019). However, optimizing combination regimens demands precision in dosing schedules, administration routes, and patient-specific factors to minimize off-target effects. Personalized approaches, guided by biomarkers linked to tumor genetics or nanoparticle uptake efficiency, could refine patient selection (Tabasum et al., 2022). Real-time monitoring via imaging or liquid biopsies may further enable adaptive treatment adjustments. Challenges such as potential interactions with systemic therapies or long-term toxicity necessitate meticulous preclinical validation to ensure compatibility and safety (Ren et al., 2024).

Overcoming manufacturing, regulatory, and integration challenges is pivotal to unlocking the potential of biogenic AgNPs for oral cancer therapy. While scalability and compliance hurdles persist, advancements in synthesis techniques, combinatorial strategies, and personalized medicine offer promising avenues. By prioritizing interdisciplinary collaboration and innovative research, AgNPs may emerge as a versatile and effective component of future cancer treatment paradigms, addressing unmet needs in precision oncology.