Integrating spatial analysis in the study of silver nanoparticles for radiotherapy: from molecular mechanisms to bio distribution in biological systems

3.1 Size and morphology

Particle size critically influences penetration, reactivity and clearance. Ultra-small AgNPs (<20 nm) penetrate cell membranes and tumor interstitium more readily and display high surface reactivity that enhances ROS generation, mitochondrial impairment and DNA damage – properties that potentiate radiosensitization. Conversely, larger AgNPs (≥50 nm) internalize more slowly, persist longer in circulation, and frequently accumulate in mononuclear phagocyte system organs (liver, spleen). Importantly, larger particles have been shown to modulate MDR mechanisms (e.g., P-gp inhibition) in some models, providing a complementary therapeutic strategy [1], 2].

3.2 Surface charge

Zeta potential affects dispersion stability, protein adsorption and cellular interactions. Positively charged AgNPs often achieve greater cellular uptake via electrostatic interactions but are more susceptible to nonspecific organ deposition; neutral or slightly negative coatings reduce opsonization and lengthen systemic half-life. Optimal surface charge balances tumor uptake with reduced off-target retention.

3.3 Surface chemistry and functional coatings

Coatings such as citrate, PEG, PVP, silica, or biogenic polyphenols regulate colloidal stability, ionic silver release, and immune recognition. PEGylation extends circulation and supports EPR-mediated passive accumulation, whereas biogenic coatings can provide intrinsic biological activity and improved biocompatibility. Drug conjugation and ligand functionalization (antibodies, peptides) add targeting and therapeutic payload capabilities, enabling theranostic strategies [3], [4], [5].

Coatings with biomolecules – such as albumin, chitosan, or gallic acid – can stabilize AgNPs, regulate ionic release, and modulate protein corona formation, which directly influences biodistribution and cellular uptake. Biogenic coatings derived from plant extracts offer an additional advantage by combining structural stability with biological functionality. Plant-based or microbial synthesis not only provides an eco-friendly alternative to conventional chemical routes but also imparts surface-bound phytochemicals that may exhibit inherent antioxidant or anticancer properties. Recent work employing Ginkgo biloba, Borago officinalis, and Rubus fairholmianus extracts has produced AgNPs with improved biocompatibility and selective cytotoxicity toward cancer cells [6], [7], [8], [9].

3.4 Synthesis method matters

Physical, chemical and biogenic (green) syntheses produce particles with distinct impurity profiles and surface chemistries; these upstream differences frequently explain inter-study discrepancies. Standardized characterization (TEM/HRTEM, DLS, zeta potential, ICP-MS for metal content, and stability tests) should be required to enable reproducible comparisons.

Synthesis → Function insight. Rather than single-parameter correlations, the evidence supports a multifactorial design principle: small, neutrally coated AgNPs favor oxidative radiosensitization, while larger, surface-engineered particles favor MDR modulation and sustained delivery. The optimal design therefore depends on the desired clinical mechanism (radiosensitization vs. MDR reversal vs. combined theranostics) [1], 2].

3.5 Surface coating and functional modifications

Surface engineering is central to tuning AgNP behavior in vivo.

Citrate-coated AgNPs provide a negative surface charge that enhances colloidal stability and dispersion in biological fluids but lack intrinsic targeting capability [3].

PEGylation – the coating of nanoparticles with polyethylene glycol – remains the most established strategy to prolong systemic circulation, minimize immune recognition, and exploit the enhanced permeability and retention (EPR) effect for tumor accumulation [4], 5].

Polymeric coatings such as polyvinylpyrrolidone (PVP) further improve dispersion and are widely applied in radiosensitization studies.

Biogenic coatings, derived from plant polyphenols, chitosan, or other natural extracts, integrate colloidal stabilization with inherent biological functionality, conferring selective toxicity toward malignant cells while reducing systemic side effects [9].

Nevertheless, these biological effects are strongly context-dependent and influenced by particle size, coating stability, and the cellular model under investigation.

Functionalization with chemotherapeutic agents – including cisplatin, gemcitabine, epirubicin, and paclitaxel – creates multifunctional AgNP systems capable of synergistic action. Such conjugates enhance intracellular drug retention, promote apoptosis, and suppress multidrug resistance (MDR) through inhibition of efflux transporters [10], 11].

For instance, graphene-decorated, PEGylated AgNPs have demonstrated improved doxorubicin delivery efficiency and controlled drug release [10], while epirubicin-functionalized AgNPs achieved notably low IC₅₀ values in hepatic cancer models [12].

Bimetallic silver–selenium nanoparticles functionalized with natural antioxidants such as quercetin and gallic acid produced an 80 % reduction in lymphoma cell viability [13].

3.6 Comparative structure–function analysis of AgNPs

A comprehensive understanding of how structural parameters – such as nanoparticle size, surface coating, and functionalization – govern biological performance is essential for the rational design of AgNP-based therapeutics. Comparative analyses of recent in vitro and in vivo studies, including findings from 2025, reveal that subtle variations in these physicochemical attributes can dramatically alter nanoparticle–cell interactions, cytotoxicity, and therapeutic outcomes. This section critically evaluates the interrelationship between AgNP structural characteristics and their functional roles in cancer therapy, providing insights into how optimized design can balance efficacy and safety.

3.7 Nanoparticle size: Implications for cytotoxicity and radiosensitization

Particle size represents a fundamental parameter governing the biological and therapeutic performance of silver nanoparticles (AgNPs). Variations in size alter their physicochemical reactivity, uptake efficiency, and the resulting cellular responses, particularly reactive oxygen species (ROS) generation and mitochondrial damage, which are central to their radiosensitizing and cytotoxic effects.

Small AgNPs (<10 nm) display superior cellular penetration and rapid internalization due to their large surface-to-volume ratios. These nanoparticles efficiently produce ROS, leading to oxidative stress, mitochondrial dysfunction, and extensive DNA fragmentation, ultimately inducing apoptosis in tumor cells. Their enhanced surface reactivity is also linked to lower half-maximal inhibitory concentration (IC₅₀) values – Barcińska et al. [1] reported an IC₅₀ of 1.6 μg/mL for 2.6 nm AgNPs in pancreatic cancer models – highlighting their high potency at low concentrations.

Larger AgNPs (≥50–75 nm), though less intrinsically cytotoxic, contribute to therapy through alternative mechanisms. They exhibit slower internalization and longer systemic residence times, allowing greater interaction with cellular efflux transporters. Gopisetty et al. [2] demonstrated that larger (∼75 nm) AgNPs suppress P-glycoprotein (P-gp) activity, thereby reversing multidrug resistance (MDR) and enhancing chemotherapeutic efficacy. This suggests a complementary mechanism wherein particle size can be tuned to favor either direct oxidative damage or MDR modulation.

Beyond single-parameter effects, size-dependent synergy has been observed in functionalized systems. Kovács et al. [14] showed that AgNPs inhibit efflux pumps in MDR cancer cells, sensitizing them to drug treatment. Similarly, Miranda et al. [9] reported that AgNPs amplify cisplatin-induced oxidative stress, selectively compromising mitochondrial integrity in tumor cells. Additional studies have demonstrated that conjugating small AgNPs with chemotherapeutics such as gemcitabine or epirubicin enhances intracellular accumulation and apoptosis [11], 12]. Expanding this concept, Mittal et al. [13] developed bimetallic Ag–Se nanoparticles functionalized with quercetin and gallic acid, achieving an ∼80 % reduction in lymphoma cell viability, confirming that combined nanoscale engineering and size control can dramatically improve therapeutic efficacy.

In summary, AgNP size is a critical determinant of both cytotoxic and radiosensitizing outcomes.

Particles <20 nm → enhance ROS generation, mitochondrial damage, and DNA fragmentation (radiosensitization).

Particles ≥50 nm → modulate drug resistance through P-gp inhibition and extended circulation.

Thus, precise control of nanoparticle size allows researchers to tailor AgNPs for specific therapeutic mechanisms – favoring oxidative radiosensitization or MDR suppression – to maximize efficacy while minimizing systemic toxicity.

3.8 Surface coating: effects on biocompatibility and tumor targeting

Surface chemistry plays a decisive role in determining the biocompatibility, bio distribution, and therapeutic specificity of silver nanoparticles (AgNPs). The nature of surface coatings regulates nanoparticle stability, immune recognition, and interaction with tumor microenvironments, thereby defining their suitability for oncological applications. Citrate-coated AgNPs provide a negatively charged surface that enhances colloidal dispersion and suspension stability in biological fluids. However, because they lack specific targeting capabilities, these nanoparticles are primarily used in baseline cytotoxicity assays and proof-of-concept studies [3]. PEGylated AgNPs, coated with polyethylene glycol, exhibit extended systemic circulation and markedly reduced immunogenicity. By exploiting the enhanced permeability and retention (EPR) effect, they preferentially accumulate in tumor tissues and are widely employed in radio sensitization and imaging research [4], 15], 16]. Likewise, polymeric coatings such as polyvinylpyrrolidone (PVP) improve colloidal stability and are frequently used to ensure reproducibility in radiobiological studies. Biogenic coatings derived from natural extracts – including G. biloba and B. officinalis – represent eco-friendly synthesis approaches that yield nanoparticles with selective toxicity toward cancer cells and lower systemic side effects [7]. Such coatings can introduce polyphenolic or chitosan residues that confer antioxidant properties and modulate the tumor microenvironment. Recent evidence underscores the multifunctionality of surface-engineered AgNPs. Habiba et al. [17] demonstrated that PEGylated silver nanocrystals induced sustained ROS generation and persistent DNA damage in colorectal cancer cells in vitro. In animal models, intratumoral injection of 56 µg AgNPs followed by 10 Gy significantly suppressed tumor growth in HCT116-bearing mice compared with radiation alone. Similarly, PVP-coated AgNPs (∼130 nm) combined with 1–6 Gy radiation reduced tumor viability in triple-negative breast cancer (TNBC) while sparing non-tumorigenic breast cells.

Functional plant-based coatings also enhance radiosensitization. Gowda et al. (2018) [16] reported that gallic-acid-coated AgNPs inhibited epithelial–mesenchymal transition (EMT) in lung cancer cells, sensitizing them to radiation. Ahmed et al. (2021) [15] designed PEG-coated Au–Ag bimetallic nanoparticles that combined radiosensitization with computed-tomography (CT) imaging, illustrating their theranostic potential. Moreover, Pourshohod et al. (2022) [18] developed HER2-targeted AgNPs exhibiting high tumor selectivity and enhanced radiation response. Dhanalekshmi et al. [4] introduced core–shell Ag@SiO₂ nanoparticles with efficient ROS generation suitable for photodynamic therapy, further demonstrating the versatility of AgNP surface design.

Nevertheless, metal composition significantly affects performance. Ahmed et al. (2022) [19] observed that PEGylated gold nanoparticles outperformed AgNPs in both radiosensitizing and imaging efficacy, emphasizing that compositional optimization is essential. Complementary work by Almayahi et al. [5], 20] investigated Ag–Mn nanocomposites exposed to γ-irradiation in colorectal and hepatic cancer models, showing enhanced cytotoxicity and improved drug delivery, particularly with particles smaller than 50 nm. The addition of silica coatings reduced toxicity to normal cells by approximately 40 %, highlighting the protective advantage of surface engineering.

Despite these advances, systemic toxicity remains a challenge. Nie et al. (2025) [21] warned that even trace levels of AgNPs may induce long-term adverse systemic effects, necessitating detailed toxicological profiling. Abbasi et al. [22] further demonstrated that nanoparticle size, shape, and surface charge strongly dictate biodistribution, clearance, and safety – parameters that must be optimized for clinical translation.

Green-synthesis strategies offer safer, biocompatible alternatives. B. officinalis-mediated AgNPs and ZnO nanoparticles exhibit potent antioxidant, antibacterial, and anticancer properties [23], while G. biloba-based biosynthesized AgNPs induce apoptosis in gastric cancer cells through oxidative stress pathways [21]. Similarly, Abdellatif et al. (2023) [23] produced chitosan-coated AgNPs (AgNPs-CHI) that reduced pro-inflammatory cytokines (IL-6, TNF-α) in breast cancer cells with minimal toxicity to fibroblasts – marking a key advance toward biocompatible cancer nanotherapy.

In vivo evidence supports these findings: Liu et al. [24] showed that PVP-coated AgNPs (21 nm, −15 mV) quadrupled median survival in C6 glioma-bearing rats following 10 Gy radiation (100.5 days vs 24.5 days for radiation alone). Tumor regression and apoptosis were substantially enhanced, particularly under hypoxic conditions – scenarios typically resistant to radiotherapy [25].

In summary, surface coatings decisively modulate the biological fate of AgNPs by controlling stability, immune recognition, and tumor targeting. PEG, PVP, and biogenic layers remain leading candidates for clinical translation, offering enhanced radiosensitization, improved therapeutic selectivity, and reduced off-target toxicity. However, persistent challenges such as dose-dependent systemic accumulation, incomplete biodegradation, and inconsistent characterization standards must be addressed through rigorous in vivo validation and unified reporting protocols. Future research should focus on optimizing nanoparticle size, surface modifications, and targeting ligands, alongside rigorous in vivo validation and comprehensive safety profiling to facilitate safe and effective clinical translation (Table 2).

Table 3 given their versatile properties, AgNPs are poised for several key clinical applications:

Early-phase preclinical and clinical studies continue to validate the safety, biodistribution, and therapeutic potential of AgNPs. Ongoing optimization of their size, shape, coating, and functional payloads will be essential for clinical translation and regulatory approval.

Size-dependent cytotoxicity: Smaller AgNPs (1–10 nm) exhibit stronger cytotoxic effects, with IC₅₀ values often below 5 μg/mL, due to enhanced cellular uptake and surface reactivity [1], 2].

ROS-mediated apoptosis: AgNPs trigger oxidative stress, mitochondrial depolarization, and caspase cascade activation, ultimately leading to programmed cell death [9]. MDR modulation: Larger AgNPs (≥50 nm) have been shown to inhibit P-glycoprotein (P-gp) activity, increasing intracellular drug accumulation and reversing MDR phenotypes [26].

This comparative structure–function analysis demonstrates that AgNP performance is highly tunable via nanoscale engineering. Parameters such as size, coating, and functionalization synergistically shape cytotoxic, radiosensitizing, and therapeutic profiles. Our work provides both a visual and quantitative framework (Figures 1 and 2) to evaluate past findings and guide future AgNP design.

Figure 1:

Nanoparticle size and IC₅₀ values.

Figure 2:

Size-dependent effects of AgNPs on cancer cells.

Figure 2 presents the size-dependent effects of AgNPs on cancer cells, offering a graphical comparison of how small and large AgNPs influence oxidative stress, mitochondrial damage, and P-glycoprotein inhibition.

The article examines how AgNPs can be used to improve cancer therapies such as radiotherapy and chemotherapy. It talks about their chemical and physical features, the actions they have (such as making reactive oxygen species and triggering apoptosis) and their impact on drug-resistant cells. It states that having the right nanoparticle size, coating and functionalization can result in higher success rates and safer treatments [27].

3.9 Lack of standardized characterization

One of the persistent challenges in nanoparticle research – particularly in the biomedical application of silver nanoparticles (AgNPs) – is the absence of universally accepted standards for their physicochemical characterization. This inconsistency undermines reproducibility, comparability, and translational reliability across studies.

4.1 Oxidative stress and reactive oxygen species (ROS) generation

AgNPs are potent inducers of oxidative stress. Their high surface reactivity facilitates electron transfer reactions that generate ROS, including superoxide anions (O₂ ⁻), hydroxyl radicals (•OH), and hydrogen peroxide (H₂O₂). Elevated ROS levels overwhelm antioxidant defense systems, leading to lipid peroxidation, protein oxidation, and nucleic acid damage. This imbalance triggers apoptosis through activation of mitochondrial permeability transition pores and the intrinsic caspase cascade [26], 28].

The magnitude of ROS production is strongly size-dependent; smaller AgNPs (<20 nm) possess a higher catalytic surface area, amplifying oxidative stress and enhancing their radiosensitizing capacity [1], 2]. Coated or functionalized AgNPs may further intensify ROS effects by stabilizing surface energy states, as observed in PEG- and polyphenol-modified systems [9], 16].

4.2 Mitochondrial dysfunction and apoptotic signaling

Exposure to AgNPs results in mitochondrial membrane depolarization, cytochrome c release, and activation of caspase-3 and -9, hallmark indicators of apoptosis. Studies have shown that AgNP-treated cancer cells display increased Bax/Bcl-2 ratios and decreased ATP production, reflecting energy depletion and loss of mitochondrial integrity [29]. When combined with radiotherapy, AgNPs act synergistically, amplifying mitochondrial oxidative stress and promoting apoptosis even in radioresistant cells [30]. The generation of ROS and reactive nitrogen species (RNS) also activates stress-related kinases (JNK, p38 MAPK), further propagating apoptotic signaling [15].

4.3 DNA damage and inhibition of DNA repair pathways

AgNPs contribute to genotoxic stress both directly, by physically interacting with nuclear components, and indirectly, through ROS-mediated DNA damage. Comet and γ-H2AX assays consistently show increased double-strand breaks in AgNP-exposed cells [28]. When combined with ionizing radiation, AgNPs delay DNA repair kinetics by inhibiting homologous recombination and non-homologous end joining pathways, thereby amplifying radiosensitization [31]. The interaction of AgNPs with zinc-finger proteins and DNA polymerases further disrupts replication fidelity and repair enzyme activity, compounding genomic instability.

4.4 Modulation of cell cycle and autophagy

AgNP exposure frequently induces cell-cycle arrest at the G₂/M checkpoint – a phase particularly sensitive to radiation – thereby enhancing tumor radiosensitivity. This arrest correlates with p53 activation and increased expression of cyclin-dependent kinase inhibitors (p21, p27) [30]. Parallel to apoptotic mechanisms, AgNPs can also activate autophagic pathways, which, depending on context, either promote survival or facilitate cell death. The balance between autophagy and apoptosis appears to be dose- and coating-dependent, with PEGylated and biogenic AgNPs showing controlled activation that favors apoptotic outcomes in malignant cells.

4.5 Synergistic interaction with radiotherapy

The radiosensitizing potential of AgNPs arises from their high atomic number (Z = 47), which enhances photon absorption and secondary electron emission during irradiation. These photoelectrons and Auger electrons locally intensify ionization, generating dense ROS fields within tumor cells. When integrated into radiotherapy protocols, AgNPs increase the linear energy transfer (LET) in targeted tissues, improving tumoricidal efficacy while minimizing collateral damage to surrounding healthy tissue. Moreover, AgNPs can downregulate DNA repair proteins such as RAD51 and XRCC1, preventing recovery from radiation-induced damage [31].

4.6 Crosstalk between oxidative stress and immune modulation

Beyond direct cytotoxicity, AgNPs influence the tumor immune microenvironment. Moderate ROS levels promote immunogenic cell death (ICD) by releasing danger-associated molecular patterns (DAMPs) such as calreticulin and HMGB1, thereby enhancing dendritic-cell activation and cytotoxic T-cell responses [30]. Conversely, excessive oxidative stress can induce immunosuppression by impairing macrophage function and T-cell proliferation. Therefore, optimizing AgNP dose and coating composition is essential to achieve a balance between tumor eradication and immune preservation.

The molecular mechanisms underlying AgNP-mediated cytotoxicity and radiosensitization are multifactorial, encompassing oxidative stress, mitochondrial collapse, DNA repair inhibition, and immune modulation. These mechanisms act synergistically with radiotherapy, particularly when particle size, surface chemistry, and dosage are carefully optimized. Understanding these interlinked pathways not only provides mechanistic insight but also informs the rational design of next-generation nanotherapeutics with enhanced selectivity and minimized toxicity. Table 4 shows summary of mechanisms.

4.7 Novel contributions from 2025 research: Emerging mechanistic insights

Recent investigations in 2025 have expanded the mechanistic understanding of AgNP-induced cytotoxicity by integrating multi-metallic design, surface engineering, and green synthesis approaches. These developments represent key steps toward safer and more effective clinical translation.

Gamma-irradiated Ag–Mn nanocomposites have emerged as a novel class of multifunctional radiosensitizers exhibiting a triple-action mechanism: enhanced ROS generation, mitochondrial disruption, and inhibition of ATP-binding cassette (ABC) transporters responsible for drug resistance. In colorectal and hepatic cancer models, these nanocomposites significantly increased apoptosis while maintaining selective toxicity toward tumor tissue [21].

Silica-coated AgNPs demonstrated a reduction in nonspecific cytotoxicity by approximately 30–40 %, improving their compatibility for biomedical applications and suggesting the protective role of inorganic surface passivation [22].

Green-synthesized AgNPs, produced using plant extracts such as G. biloba and B. officinalis, exhibited remarkable selectivity toward cancer cells while maintaining biocompatibility. Their synthesis integrates natural antioxidants and polyphenols, which both stabilize nanoparticle surfaces and reduce oxidative side effects, offering a sustainable and low-cost route to scalable nanomedicine production [23].

Collectively, these findings outline a mechanistic framework linking nanomaterial design to therapeutic response, emphasizing that controlled irradiation, biogenic synthesis, and hybrid composition can fine-tune AgNP performance for precision oncology.

Figure 3 depicts the mechanisms of AgNP interaction with cancer cells, illustrating how AgNPs penetrate the cells, generate ROS, impair mitochondrial function, and suppress drug resistance proteins.

Figure 3:

Mechanisms of AgNP interaction with cancer cells.

Illustration showing key cellular pathways affected by AgNP exposure: 1) nanoparticle penetration through the plasma membrane; 2) generation of reactive oxygen species (ROS); 3) mitochondrial membrane depolarization leading to cytochrome c release; 4) nuclear DNA fragmentation; and 5) suppression of drug-resistance proteins such as P-glycoprotein (P-gp).

AgNP-induced cytotoxicity arises primarily from ROS-mediated mitochondrial and DNA damage, with magnitude dependent on nanoparticle composition, size, and coating. Standardized experimental models are required to quantify these relationships and establish reproducible therapeutic predictions. Mechanistic insights from recent 2025 research underscore how engineered AgNPs – especially γ-irradiated and biogenic variants – can synergize with chemotherapy and radiotherapy to improve selectivity and treatment efficacy. The next section explores how these molecular principles translate into in vivo outcomes, highlighting biodistribution, pharmacokinetics, and toxicity patterns of AgNP-based therapeutic systems.

5.1 Biodistribution patterns

Following administration, AgNPs distribute rapidly across multiple organs due to their nanoscale dimensions and dynamic surface chemistry. Accumulation predominantly occurs in the liver, spleen, lungs, and kidneys – organs associated with the mononuclear phagocyte system (MPS). Particle size plays a decisive role in organ targeting: Small AgNPs (<20 nm) exhibit enhanced vascular permeability and tumor accumulation through the enhanced permeability and retention (EPR) effect but are also prone to rapid renal clearance. Intermediate-sized AgNPs (20–50 nm) maintain a balance between circulation time and tissue penetration, often demonstrating optimal tumor retention. Larger AgNPs (>50 nm) have extended half-lives but preferentially accumulate in hepatic and splenic tissues, reflecting phagocytic sequestration. Surface coatings profoundly influence biodistribution. PEGylation and PVP coatings extend circulation and reduce uptake by the MPS, allowing higher tumor accumulation [15], 16]. Conversely, uncoated or positively charged AgNPs interact strongly with plasma proteins, forming a thick protein corona that enhances opsonization and clearance. Biogenic or chitosan coatings can improve biocompatibility while promoting selective tumor uptake, as demonstrated in multiple in vivo models [23], 24].

5.2 Pharmacokinetics and clearance

AgNP pharmacokinetics are typically biphasic – an initial rapid distribution phase followed by a slower elimination phase. Renal and hepatobiliary pathways represent the primary routes of excretion, with smaller particles (<10 nm) efficiently filtered by the glomerulus, whereas larger or aggregated particles undergo biliary clearance. Studies employing inductively coupled plasma mass spectrometry (ICP-MS) have confirmed persistent silver residues in liver and spleen up to several weeks post-injection, suggesting incomplete clearance of larger particles. PEGylated and silica-coated formulations have been shown to improve renal excretion and reduce hepatic retention, enhancing long-term biocompatibility.

5.3 Blood circulation and protein corona formation

Once introduced into systemic circulation, AgNPs interact with plasma proteins to form a “protein corona,” which fundamentally alters their biological identity. The composition of this corona depends on particle size, charge, and surface chemistry and dictates biodistribution, cellular uptake, and immunogenicity. PEG and PVP coatings reduce nonspecific protein adsorption, extending circulation time, while biogenic coatings rich in polyphenols may favor selective binding to cancer-associated proteins. Understanding corona dynamics is therefore essential for designing AgNPs with predictable in vivo behavior.

5.4 Toxicological considerations

While AgNPs show strong anticancer potential, their safety profile remains a significant concern. Systemic accumulation of ionic silver and long-term retention in MPS organs can trigger inflammation, oxidative stress, and organ dysfunction. Histopathological studies have documented dose-dependent alterations in hepatic enzyme activity, nephrotoxicity, and pulmonary inflammation at high concentrations [21], 22]. The release of Ag⁺ ions from unstable nanoparticles contributes substantially to these effects, underscoring the need for stable coatings that minimize dissolution.

Green-synthesized and silica-coated AgNPs display improved safety, with lower oxidative and inflammatory responses compared to chemically reduced nanoparticles [7], 23]. Abdellatif et al. [23] reported that chitosan-coated AgNPs reduced pro-inflammatory cytokine expression (IL-6, TNF-α) in breast cancer models without significant cytotoxicity to fibroblasts. Nevertheless, Nie et al. (2025) [21] cautioned that even trace AgNP exposure could induce chronic systemic effects, reinforcing the necessity of comprehensive toxicological assessments before clinical application.

5.5 Tumor selectivity and therapeutic index

AgNPs exploit both passive and active mechanisms for tumor accumulation. Passive targeting arises from the EPR effect, while active targeting is achieved through ligand conjugation (e.g., antibodies or peptides) that recognize tumor-specific receptors. Optimal designs balance effective tumor localization with rapid clearance from healthy organs. Recent comparative studies indicate that AgNPs smaller than 30 nm, coated with PEG or silica, provide the best combination of tumor selectivity and systemic safety [5], 20]. This design principle supports their continued development for combined radiotherapy and chemotherapy applications.

AgNP biodistribution and pharmacokinetics are intricately linked to their structural and surface properties. Smaller PEG- or biogenic-coated particles favor tumor penetration and renal clearance, while larger uncoated forms tend to accumulate in MPS organs, increasing the risk of chronic toxicity. Standardized, quantitative in vivo assessments of AgNP absorption, distribution, metabolism, and excretion (ADME) are urgently needed to ensure reproducible and clinically translatable results. Rational design – emphasizing controlled size, stable coatings, and green synthesis – remains the cornerstone of developing safe and effective AgNP-based radiotherapeutics.

6.1 Radiosensitization: enhancing radiotherapy outcomes

AgNPs significantly improve the efficacy of ionizing radiation by acting as potent radiosensitizers, especially in hypoxic and radiation-resistant tumors. Their relatively high atomic number (Z = 47) contributes to enhanced photoelectric absorption and emission of secondary electrons during radiotherapy, which results in:

Localized dose enhancement, particularly near tumor cell DNA.

Excessive ROS generation, amplifying oxidative stress and double-strand DNA breaks [33].

Apoptotic signaling cascade activation, including mitochondrial disruption and caspase activation [15], 30]. In vivo studies have demonstrated improved tumor control and prolonged survival in glioblastoma and colorectal cancer models treated with AgNPs in combination with radiation [24].

6.2 Chemotherapy synergy and reversal of multidrug resistance (MDR)

MDR is a critical barrier to successful cancer treatment. It is commonly mediated by the overexpression of ATP-binding cassette (ABC) transporters, particularly P-glycoprotein (P-gp), which actively expel chemotherapeutic drugs from cancer cells [32], 33]. This efflux mechanism diminishes intracellular drug concentration, resulting in decreased sensitivity to treatment. MDR is prevalent across multiple cancer types, including breast, lung, and colon cancers, and is strongly associated with disease recurrence and poor prognosis.

AgNPs enhance the potency of conventional chemotherapeutic agents via several mechanisms:

Inhibition of drug efflux pumps, such as P-glycoprotein (P-gp), leading to increased intracellular retention of drugs [26]. Disruption of MDR-associated pathways, restoring sensitivity to agents like cisplatin, paclitaxel, and gemcitabine [10], 11]. Co-amplification of oxidative and apoptotic stress, especially when combined with mitochondrial-targeting drugs. For instance, AgNP–cisplatin conjugates have been shown to sensitize resistant cancer cells while minimizing off-target cytotoxicity, suggesting a potential role in dose reduction strategies [28]. One of the major challenges in cancer treatment is MDR, which often arises due to the overexpression of drug efflux transporters such as P-glycoprotein (P-gp). AgNPs have been shown to reverse MDR through several mechanisms: Inhibiting the activity or expression of efflux pumps, thereby allowing chemotherapeutic agents to accumulate within cancer cells. Modulating cellular signaling pathways associated with drug resistance, including PI3K/Akt, MAPK, and NF-κB pathways. Inducing oxidative stress and mitochondrial dysfunction that sensitize resistant cells to chemotherapy. Studies by Li et al. (2016) and Karuppaiah et al. (2020) [11], 28] provide compelling evidence that AgNPs can sensitize resistant cancer cell lines to chemotherapeutic agents, restoring their susceptibility and enhancing overall treatment efficacy.

6.3 Targeted drug delivery

AgNPs can be surface-modified with various biological ligands to achieve tumor-specific delivery, improving therapeutic precision and reducing systemic side effects. Key strategies include:

PEGylation: Increases circulation time and tumor accumulation via the Enhanced Permeability and Retention (EPR) effect. Antibody or ligand conjugation: Enables receptor-mediated endocytosis (HER2-targeted AgNPs in breast cancer). Biogenic or polymeric coatings: Enhance biocompatibility, immune evasion, and payload protection. Such modifications result in enhanced therapeutic indices, allowing higher drug concentrations at tumor sites with reduced collateral toxicity.

6.4 Photothermal and photodynamic therapies (PTT/PDT)

AgNPs are increasingly explored in light-triggered cancer therapies due to their tunable optical properties and catalytic activity:

Photothermal Therapy (PTT): AgNPs absorb near-infrared (NIR) light and convert it into heat, inducing localized hyperthermia that selectively ablates tumor cells.

Photodynamic Therapy (PDT): AgNPs facilitate singlet oxygen and ROS generation upon light activation, leading to oxidative damage of cellular components [18].

AgNPs integrated into core–shell architectures (Ag@SiO₂) or conjugated with photosensitizers have demonstrated enhanced efficacy in various cancers, including cervical, breast, and head-and-neck malignancies [20].

6.5 Multimodal and theranostic integration

The next frontier in AgNP-based cancer therapy lies in multimodal integration, where AgNPs are simultaneously employed for therapy, targeting, and imaging. Such multifunctional platforms can enable:

Radiosensitization + chemotherapy enhancement

Targeted delivery + photothermal ablation

Real-time imaging for treatment monitoring (theranostics)

By integrating therapeutic and diagnostic functionalities into a single nanosystem, AgNPs provide a synergistic and personalized approach to oncology, capable of improving treatment specificity and outcomes.

AgNPs offer promising capabilities in multimodal cancer therapy, enabling combinations of:

Radiosensitization (via ROS and secondary electron emission)

Chemotherapy enhancement (through drug conjugation and MDR inhibition)

Photodynamic and photothermal therapy (PDT/PTT)

Imaging (CT or fluorescence contrast enhancement)

This integrative strategy aligns with the goals of precision oncology, facilitating real-time tracking and personalized treatment regimens.

6.6 Clinical potential and translational opportunities

AgNPs have emerged as potent radiosensitizers capable of enhancing the efficacy of radiotherapy (RT) by increasing cancer cell susceptibility to ionizing radiation. This radiosensitizing effect is primarily achieved through two key mechanisms:

Enhanced DNA Damage: AgNPs amplify radiation-induced DNA double-strand breaks (DSBs), a critical form of cytotoxic damage, by emitting secondary electrons when exposed to X-rays or γ-rays.

Increased ROS: These secondary electrons also catalyze ROS production via water radiolysis, causing oxidative stress, mitochondrial dysfunction, lipid peroxidation, and endoplasmic reticulum (ER) stress – ultimately leading to apoptosis and autophagic cell death.

Due to their high atomic number (Z = 47), AgNPs possess a dense electron cloud that enhances their interaction with radiation, similar to other high-Z radiosensitizers like gold and bismuth [15], 16], 28]. The emitted low-energy electrons and ROS generated by AgNPs increase localized radiation doses within tumors, while sparing adjacent healthy tissues [34], 35].

6.6.2 Enhancement of chemotherapeutic efficacy

AgNPs have demonstrated promising synergistic effects when used in combination with conventional chemotherapeutic agents such as cisplatin and paclitaxel. These combinations result in significantly enhanced cytotoxicity against cancer cells. AgNPs can facilitate increased cellular uptake of chemotherapeutic drugs by altering membrane permeability and endocytic pathways, thereby improving intracellular drug concentrations and therapeutic response.

In addition to enhancing chemotherapy, AgNPs serve as potent radiosensitizers. Their ability to generate ROS, induce DNA double-strand breaks, and disrupt key survival pathways further amplifies the cytotoxic effects of ionizing radiation. The dual-action of AgNPs – potentiating both radiotherapy and chemotherapy – offers a highly synergistic approach to combating resistant and aggressive tumors.

6.7 Summary of key trends and implications

Cumulative preclinical evidence highlights the strong anticancer potential of AgNPs through mechanisms including ROS generation, mitochondrial dysfunction, and modulation of drug resistance pathways. The advent of multifunctional nanocomposites and green synthesis approaches has further enhanced safety and efficacy profiles. Notably, the 2025 studies by Almayahi et al. represent a pivotal advancement – the first documented use of gamma-irradiated Ag–Mn nanocomposites for dual-action targeting via mitochondrial disruption and ABC transporter inhibition, offering a promising strategy for overcoming therapeutic resistance in cancer. Figure 4 illustrates the interaction of AgNPs within biological systems, highlighting cellular uptake pathways and intracellular effects.

Figure 4:

Bio distribution and spatial analysis of silver nanoparticles in human organs.

6.8 Therapeutic functionalization: enhancing anticancer efficacy

Functionalization of AgNPs with chemotherapeutic agents enables synergistic effects through improved targeting, drug delivery, and apoptosis induction:

AgNP–Cisplatin: Amplified DNA damage and ROS production in hepatocarcinoma models [9].

AgNP–Paclitaxel: Enhanced intracellular delivery and ROS-mediated apoptosis in liver cancer [28]. AgNP–Gemcitabine: Achieved potent synergistic cytotoxicity in metastatic breast cancer [11]. Ag–Se bimetallic nanoparticles: Simultaneously activated oxidative and apoptotic pathways, reducing lymphoma viability by 80 % [13]. Understanding these structural parameters provides the basis for exploring how AgNPs modulate intracellular pathways, as discussed in the next section. Overall, AgNPs act as synergistic enhancers across radiation, chemotherapy, and phototherapy modalities. Their clinical promise lies in designing multifunctional systems that combine controlled release, imaging visibility, and minimized systemic toxicity. While therapeutic outcomes strongly depend on the intrinsic physicochemical and mechanistic properties of AgNPs, their real effectiveness in vivo is ultimately determined by how these nanoparticles distribute, accumulate, and clear within biological systems. Understanding this spatial biodistribution is critical for predicting treatment efficacy, minimizing off-target toxicity, and refining nanoparticle design. Advanced imaging and analytical tools now make it possible to visualize and quantify AgNP localization at the organ, tissue, and even cellular levels. The following section examines these imaging-based approaches and their role in translating AgNP research from experimental settings to clinically meaningful applications.

7.1 Imaging and mapping techniques

PET and SPECT: Radiolabeled AgNP tracking in vivo.

MRI: For hybrid AgNP-based contrast agents.

Fluorescence/Bioluminescence imaging: For small-animal studies.

ICP-MS and LA-ICP-MS: Quantitative tissue analysis and spatial mapping.

Hyperspectral imaging: Visualizing AgNPs in cells/tissues.

7.2 Factors influencing spatial distribution

Targeted AgNPs show 3–5 × higher tumor uptakes than uncoated forms.

Liver, spleen, kidneys, and lungs are common accumulation sites.

Small AgNPs (<10 nm) are cleared renally; larger rely on hepatobiliary excretion.

Regions with high AgNP density correlate with greater radiosensitization.

Figure 5 shows AgNP injection, biodistribution routes (tumor, liver, spleen, kidney), imaging modalities used (PET, SPECT, ICP-MS), and comparative heat maps for targeted versus non-targeted nanoparticles.

Figure 5:

Schematic representation of AgNP injection, bio distribution routes, imaging modalities used (PET, SPECT, ICP-MS), and comparative heat maps for targeted versus non-targeted nanoparticles.

7.3 Strategies to improve spatial targeting

Ligand functionalization (HER2, EGFR targeting).

Stimuli-responsive coatings (pH, glutathione-sensitive).

Magnetic guidance systems.

Pre-targeting approaches for improved specificity.

7.4 Challenges and future prospects

Remaining issues include patient variability, limited clinical imaging infrastructure, and lack of standardized quantification protocols. Future directions involve combining real-time imaging, computational modeling, and quantitative tissue mapping to design AgNPs with predictable biodistribution and optimal therapeutic profiles.

Accurate spatial quantification of AgNP biodistribution bridges the gap between molecular mechanism and therapeutic performance. Future research should employ multimodal imaging and standardized reporting metrics to compare outcomes across models and accelerate clinical translation.

Integrating spatial imaging data with mechanistic understanding provides a multidimensional perspective on AgNP behavior in biological systems. However, translating these findings into reliable therapeutic strategies requires validation through systematic preclinical experimentation. Comprehensive in vitro and in vivo studies allow assessment of how particle characteristics, biodistribution profiles, and radiation or drug interactions collectively influence treatment outcomes. The following section critically evaluates this preclinical evidence, emphasizing both the demonstrated therapeutic benefits and the methodological limitations that must be addressed before advancing to clinical translation.

8.1 Clinical potential and research outlook

While preclinical studies demonstrate the multifaceted therapeutic benefits of AgNPs, further investigations are necessary to elucidate their precise molecular mechanisms, optimize dosage, and assess long-term safety. Comprehensive clinical trials are essential to validate their translational potential in oncology. Table 6 shows in vitro studies (2018–2022) evaluating the radiosensitizing efficacy of AgNPs across various cancer cell lines.

8.2 Preclinical evaluations and experimental evidence

Over the past decade, a growing body of in vitro and in vivo studies has explored the biological behavior, cytotoxic mechanisms, and radiosensitizing capabilities of AgNPs across various cancer models. These investigations provide a robust foundation for the translational development of AgNPs as promising adjuncts in cancer therapy.

8.3 In vitro studies: anticancer mechanisms and efficacy

Numerous in vitro studies have assessed the anticancer effects of AgNPs against a range of human cancer cell lines, including:

Hepatocellular carcinoma (HepG2)

Breast adenocarcinoma (MCF-7)

Cervical cancer (HeLa)

Pancreatic carcinoma (PANC-1)

Lung adenocarcinoma (A549)

Colorectal carcinoma (HTC-116)

8.4 In vivo studies: tumor inhibition and radiosensitization

In vivo animal models further validate the therapeutic potential of AgNPs in cancer treatment and radiosensitization:

In colorectal tumor-bearing mice, intratumoral injection of PEGylated AgNPs followed by a 10 Gy radiation dose significantly reduced tumor volume and prolonged survival [16]. In glioma-bearing rats, PVP-coated AgNPs enhanced apoptosis and survival rates under both normoxic and hypoxic conditions, demonstrating efficacy even in radioresistant tumor environments [20]. These studies support the role of AgNPs as potent radiosensitizers capable of enhancing radiotherapeutic outcomes, especially under conditions that typically reduce radiation efficacy.

8.5 Recent advances and experimental findings (2025)

Emerging research in 2025 has introduced novel AgNP-based nanocomposites and eco-friendly synthesis methods with improved therapeutic indices:

Ag–Mn Nanocomposites (Almayahi et al. 2025) [5], 20]:

Gamma-irradiated Ag–Mn nanoparticles displayed enhanced cytotoxicity through mitochondrial disruption and ATP-binding cassette (ABC) transporter inhibition. In colorectal and liver cancer models, IC₅₀ values ranged from 21–42 μg/mL, depending on particle size and surface coating. Encapsulation with a silica shell improved safety by reducing toxicity to normal cells by 30–40 %, indicating a significant improvement in biocompatibility.

Phytogenic AgNPs: Green synthesis using B. officinalis and G. biloba extracts produced nanoparticles with selective cytotoxicity, strong antioxidant activity, and broad-spectrum antimicrobial properties [44]. These AgNPs induced apoptosis in cancer cells while exhibiting minimal effects on healthy tissues, supporting their potential for safe, targeted cancer therapy.

8.6 Comparative data analysis: In vitro and in vivo profiles

To support rational design and clinical translation, Tables 5 and 6 provide a comparative overview of:

Table 5:

Summary of experimental in vitro studies (2014–2024) evaluating the anticancer activity of AgNPs.

Ref.	Size (nm) and ZP (mV)	Coated or doped- surface modifier-conjugates	Concentration μg/mL; treatment duration	Cell line	Characterizations	IC50/viability%	Effects observed
Miranda et al. [9]	10; −38.9 ± 1.75	Citrate	0–10, 24 h	Hepatocarcinoma cells (HepG2) THLE2	TEM, ICP–MS, DLS and ZP, FLUOstar	Viability: HepG2 = 25 %, THLE2 = 70 %	Selective cytotoxicity in cancer cells, apoptosis induction, oxidative stress
Barcinska et al. [1]	1–5 (2.6 ± 0.8); 10–26 (18 ± 2.6)	NA	0.5–100; 24 h	Pancreatic cancer (Panc-1)	TEM with EDS; SensiFAST PCR	IC50: 2.6 nm = 1.6 μg/mL, 18 nm = 26.8 μg/mL	Smaller AgNPs induce stronger ROS, apoptosis; size-dependent cytotoxicity
Miranda et al. [3]	10; −30. 1 ± 3.28	Citrate	3.5; 24 h	Hepatocarcinoma (HepG2)	TEM, LC-MS/MS	Viability: 75 %	Induced metabolic adaptation, reduced proliferation
Kovács et al. [14]	28; −44	Citrate	20-100; 24 h	Adenocarcinoma (Colo 205 and Colo 320)	TEM, zeta potential, SEM	IC50: Colo 205 = 49.6 μM, Colo 320 = 58.4 μM	DNA fragmentation, mitochondrial dysfunction, apoptosis
Gopisetty et al. [2]	5 and 75, NA	Citrate	150 μM; 24 h, 48 h	Breast adenocarcinoma (MCF)	TEM, ICP-MS	IC50: 5 nm = 244 μM, 75 nm = 414 μM	5 nm AgNPs cause more oxidative stress; 75 nm inhibit P-glycoprotein (Pgp)
Miranda et al. [29]	1–2; −23	Cadmium and mercury	0.35, 3.5; 24 h	Hepatocarcinoma (HepG2)	ICP-MS, zeta potential, TEM, UV–vis, DLS	Viability: 80 %	Increased ROS, impaired antioxidant defense
Krzyzanowski et al. 2021 [26]	20; NA	NA	25–50;1–24 h	Liver and lung adenocarcinoma cells (HepG2 and A549)	PCR gene expression analysis, spectrophotometery EnVision multilabel reader	IC50 (72 h): HepG2 = 15.8 μg/cm3, A549 = 202.7 μg/cm3	HepG2 more sensitive, ABC transporter inhibition, apoptosis
Xu et al. [7]	40, −34.5	Biogenic (Ginkgo biloba leaves extract)	3, 6; 12–36 h	Cervical adenocarcinoma cells (HeLa)	UV–vis, TEM, DLS, zeta-potential	Viability: 4 μg/mL = 30 %	Mitochondrial pathway apoptosis, high ROS generation
George et al., [8]	30–150; −24.5	Biogenic (Rubus fairholmianus)	2.5–10; 24 h	Breast adenocarcinoma (MCF-7)	XRD, UV–vis, FTIR, SEM, TEM, zeta potential	NA	Caspase activation, increased Bax/P53 expression, apoptosis
Rozalen et al. 2020 [30]	14.7 (DLS), 11.13 (HRTEM)	NaBH4 , trisodium citrate dehydrate , NaOH	38-760;12–48 h	Colorectal cancer (HTC-116)	XRD, UV–vis, FTIR, TEM, zeta potential	IC50: 186 μg/mL (12 h), 98 μg/mL (24 h), 63 μg/mL (48 h)	Dose-dependent cytotoxicity, mitochondrial depolarization
Rozalen et al. 2020 [30]	21.9 (DLS), 15.20 (HRTEM)	MTX	38-760;12–48 h	Human lung carcinoma (A-549)	XRD, UV–vis; FTIR, TEM, zeta potential	IC50: 88 μg/mL(12 h) 38 μg/mL(24 h) 23 μg/mL(48 h)	Lower sensitivity and slower response time; no significant effect of AgNPs alone; AgNPs-MTX showed some cytotoxic activity​
Palai et al. [10]	25 (HRTEM)	mPEG-NH2 DOX	1–100; 48 h	Cervical adenocarcinoma (HeLa)	XRD, UV–vis, FTIR, HRTEM, zeta potential	Viability: 41.56 %	Enhanced drug delivery, reduced multidrug resistance (MDR)
Ding et al. [12]	36(TEM)	Epirubicin	0-30; 48 h	Liver cancer (HepG2)	UV-vis, FTIR,TEM, EDX	IC50: 1.92 μM	DNA damage, G2/M phase cell cycle arrest
Li et al. 2016 [32]	TEM: >2	Polyethylenimineand paclitaxel	2.5; 24 h	Liver cancer (HepG2)	TEM, zeta potential	Viability: 58.32 %	Induced HepG2 cell apoptosis via mitochondrial dysfunction, ROS generation, caspase-3 activation, and p53/MAPK/AKT signaling pathways​
Karuppaiah et al. [11]	>25(SEM, TEM): 9.16 (DLS)	Gemcitabine , polyvinyl pyrrolidone	1.56–100 μM; 24 h	Metastatic breast cancer (MDA-MB-453)	UV–vis, SEM, EDX, TEM, zeta potential	Viability: 37.64 %	Synergistic cytotoxicity with GEM, ROS-mediated apoptosis
Mittal et al. [13]	30–35	Quercetin – Gallic acid + Se	50–500 μg/mL	Dalton lymphoma (DL)	UV–vis, TEM, FTIR, zeta potential, XRD, EDX	Viability: 20 % at 50 μg/mL	Synthesized bimetallic (Ag–Se) nanoparticles using quercetin and gallic acid; observed 80 % reduction in DL cell viability at 50 μg/mL

1. TEM, transmission electron microscope; ICP-MS, inductively coupled plasma-mass spectrometry; DLS, dynamic light scattering; ZP, zeta potential; FLUOstar Omega Plate Reader; SensiFAST SYBR No-ROX PCR Master Mix; LC-MS/MS, liquid chromatography-tandem mass spectrometry analysis; XRD, X-ray diffraction; FTIR, fourier transform infrared spectroscopy; SEM, scanning electron microscope; EDX, energy-dispersive X-ray spectroscopy (EDX or EDS analysis); UV, ultraviolet-visible spectroscopy; LC-MS-MS, liquid chromatography with tandem mass spectrometry; NA, data not available.

Table 6:

Summarizes recent in vitro studies (2018–2022) evaluating the radiosensitizing efficacy of AgNPs across various cancer cell lines.

Ref.	Size (nm) and ZP (mV)	Coated or doped- surface modifier-conjugates	Concentration μg/mL; treatment duration	Cell line	Analysis devices	RT dose and exposure duration	Viability/survival rate (%)	Effects observed
Gowda et al. 2018 [16]	10 to 30	Gallic acid	5–200 μg/ml	Lung cancer (A549-rabbit)	UV, TEM, zeta potential, FTIR, q-PCR inverted phase contrast microscope	8Gy(X-ray)	Survival: 46.5 μg/mL	Inhibited EMT, reduced metastasis, improved radiosensitization
Ahmed et al. 2021 [15]	50 ± 5 nm (DLS: −5 m)	PEG-coated Au–Ag alloy	0.01–0.34, 24 h	KB oral cancer	TEM, zeta potential, SEM, EDX, CT, confocal microscopy, NMR, Raman spectroscopy, ICP-MS	X-rays: 0, 2, 4, 6, 8 Gy	Viability: 55 %	Enhanced radiosensitization, apoptosis via ROS generation
Pourshohod et al. 2022 [18]	120–130 nm (DLS)	Cysteamine, citrate, HER2	3.9–125 μM, 72 h	Breast cancer (SK-BR-3, MCF-7, head and neck cancer (HN-5), ovarian cancer (SK-OV-3)	AFM,Zeta potential, DLS, UV–vis	X-rays: 10 Gy	Survival: 77.14 % (SK-BR-3), 55.32 % (MCF-7), 38.28 % (HN-5), 31.30 % (SK-OV-3)	HER2 targeting improved radiosensitivity, induced apoptosis
Dhanalekshmi et al. [4]	20–40 nm	SiO2	50–200 μg/ml, 16 h	Cervical cancer (HeLa)	UV–vis, XRD, FTIR, TEM, EDX	LED light: (410 nm. 84.75–34.97 J/cm2)	IC50: 119.64 μg/mL (high dose), 48.95 μg/mL (low dose)	PDT-enhanced cytotoxicity, ROS-mediated cell death
Ahmed et al. 2022 [19]	∼50 ± 5 nm, 18 Mv	PEG-600 (polyethylene glycol)	0.007–0.23 μg/ml, 24 h	Oral cancer (KB cells)	NMR, ICP-MS, UV–vis, TEM, Zeta potential, DLS	X-ray: 0, 2, 4, 6, and 8 Gy),	Viability: 70 %	Improved CT contrast, radiosensitization, apoptosis induction

Cytotoxic profiles across different cancer types

Influence of size, surface chemistry, and synthesis method on selectivity and therapeutic efficacy.

Radiosensitization outcomes, including dose–response relationships and tumor microenvironment adaptation.

This integrative approach facilitates identification of optimal AgNP formulations tailored for specific cancer types and therapeutic combinations.

Understanding these structural parameters provides the basis for exploring how AgNPs modulate intracellular pathways, as discussed in the next section.

Overall, the accumulated evidence supports the therapeutic potential of AgNPs but also underscores the need for standardized protocols and integrated imaging to validate consistency between experimental systems and clinical expectations.

Although preclinical investigations consistently demonstrate the therapeutic promise of AgNPs, they also reveal critical gaps that limit direct clinical translation. Variability in synthesis, dosing, and study design often complicates comparison across experimental models, and comprehensive long-term safety evaluations remain scarce. Understanding these challenges is essential for developing reliable, reproducible, and ethically sound nanomedicine protocols. The next section addresses these limitations, focusing on biosafety concerns, regulatory considerations, and the steps needed to transform AgNP research into clinically applicable cancer therapies.

9.1 Cytotoxicity to normal tissues

Although AgNPs are designed for selective toxicity against cancer cells, they frequently accumulate in non-target organs such as the liver, kidneys, and spleen. This unintended deposition may induce inflammation, oxidative stress, and chronic organ dysfunction, particularly when the nanoparticles are non-biodegradable.

9.2 Off-target effects

The unspecific uptake by healthy cells can trigger cytotoxic cascades similar to those observed in cancer cells, compromising tissue integrity and leading to long-term adverse effects.

9.3 Long-term toxicity and environmental risks

9.4 Environmental impact

Beyond direct human health implications, the widespread use of AgNPs poses environmental risks. Nanoparticle runoff and bioaccumulation can disrupt aquatic ecosystems, potentially leading to adverse outcomes in non-target organisms.

9.5 Regulatory and ethical barriers

9.6 Ambiguity in regulatory classification

AgNPs – and particularly novel multifunctional or hybrid formulations – often fall into a regulatory gray zone (e.g., drug, device, or biologic), creating additional challenges in classification and approval processes.

9.7 Limited long-term data

The scarcity of comprehensive animal and human studies on chronic exposure further hampers the development of clear regulatory guidelines, necessitating further research to ensure safety and efficacy before widespread clinical implementation.

Addressing safety and regulatory challenges is the final prerequisite to unlocking the clinical potential of AgNP-based nanomedicine. Once standardized synthesis, reproducible characterization, and comprehensive toxicity assessments are achieved, AgNPs can move from experimental platforms toward approved medical applications. Building on the preceding discussion of limitations and translation hurdles, the following concluding section summarizes the integrated findings of this review and outlines future research directions required to ensure the safe, effective, and targeted use of AgNPs in cancer therapy.