Evaluating the mechanisms and therapeutic potential of ZnO nanoparticles as selective anticancer agents for lung malignancies

Abstract

Lung cancer's high mortality necessitates refined, targeted therapeutic interventions. Zinc oxide nanoparticles (ZnO NPs) have emerged as promising anticancer agents owing to their distinctive physicochemical characteristics, particularly their pH-dependent solubility, and their preferential toxicity toward malignant cells. This review critically examines the mechanisms, advantages, and limitations of ZnO NPs as a novel therapeutic strategy for lung cancer. The primary antitumor mechanism involves the generation of reactive oxygen species (ROS), which disrupts the cellular redox balance and induces apoptosis. ZnO NPs are shown to trigger apoptosis by compromising mitochondrial integrity, activating caspase cascades, and altering the expression of Bax and Bcl-2 proteins. Furthermore, they impede cancer cell growth by enforcing a G2/M cell cycle arrest. Selectivity is achieved via the enhanced permeation and retention (EPR) effect and electrostatic affinity, wherein their positive surface charge (at physiological pH) promotes binding to anionic cancer cell membranes. Despite these advantages, significant challenges in biocompatibility, long-term toxicity, and in vivo stability must be addressed to facilitate clinical translation. This review underscores the critical need for further research to unlock the full therapeutic potential of ZnO NPs.

Introduction

As one of the foremost causes of cancer mortality worldwide, lung cancer is attributed to approximately 1.8 million deaths annually [1–3]. The latest global data (2022 estimates) underscore the scale of this challenge, with lung cancer accounting for 12.4% of all new cancer diagnoses and 18.7% of all cancer-related deaths [4] (Fig. 1). As illustrated in Fig. 1, lung cancer is unique in its global impact, leading all other cancers in both new diagnoses (2.5 million cases) and mortality (1.8 million deaths).

Fig. 1.

Global lung cancer epidemiology in 2022. This infographic summarizes worldwide lung cancer statistics based on the latest IARC GLOBOCAN 2022 estimates, which were released in February 2024.. The reported data includes 2.5 million newly diagnosed cancer cases, constituting 12.4% of all diagnoses, and 1.8 million cancer-related deaths, representing 18.7% of global cancer fatalities. Lung cancer is both the most frequently diagnosed cancer and the primary cause of cancer-related deaths worldwide. Incidence rate comparisons highlight disparities by region, with the highest in Australia/New Zealand (507.9 per 100,000) and the lowest in West Africa (97.1 per 100,000)

Despite considerable progress in surgical procedures and treatments such as chemotherapy and immunotherapy, the prognosis for patients—particularly those at an advanced stage—remains alarmingly unfavorable [3, 4]. The limitations of conventional therapies, including systemic toxicity and the development of drug resistance, emphasize the critical need for new, more effective, and precisely targeted therapeutic strategies for different cancers especially lung cancer [5–9]. Among the most innovative approaches emerging from the intersection of oncology and nanotechnology is the application of nanoparticles, especially zinc oxide nanoparticles (ZnO-NPs), in the diagnosis and treatment of lung cancer [10, 11].

Nanotechnology has opened new avenues for precision medicine, presenting versatile systems that can be engineered for the regulation of controlled drug release, specific tumor targeting, enhanced imaging, and reduced systemic toxicity [12–14]. In this domain, ZnO-NPs have drawn considerable focus owing to their exceptional physicochemical properties and biological activities. Key attributes such as an elevated surface area-to-volume ratio, photoluminescence, biocompatibility, and the capacity to generate reactive oxygen species (ROS) under certain conditions, make these nanoparticles suitable for inducing targeted cytotoxicity in cancer cells while showing preferential toxicity against cancer cells [15, 16]. By modifying their surface with targeting ligands, antibodies, or polymers, these nanoparticles can be optimized for increased tumor specificity and improved biodistribution, making them flexible platforms for a wide range of therapeutic purposes [17].

While ZnO NPs share anticancer properties with other metal oxide nanomaterials like iron oxide (Fe₃O₄) or titanium dioxide (TiO₂), they possess a unique set of advantages and limitations that set them apart. A primary advantage is their inherent "smart" drug release mechanism; unlike highly stable gold (Au) or iron oxide NPs, ZnO NPs exhibit significant pH-dependent solubility [18, 19]. This allows them to remain relatively inert at physiological pH but rapidly dissolve and release a flood of cytotoxic Zn²⁺ ions specifically within the acidic tumor microenvironment, a "Trojan horse" effect [20]. Furthermore, zinc is an essential trace element, suggesting a superior biocompatibility profile compared to materials like silver (Ag) NPs, as the body has endogenous mechanisms for zinc homeostasis [21]. However, this same dissolution property is also their chief limitation. Premature dissolution in the bloodstream can lead to systemic toxicity, a challenge in stability that more inert nanomaterials like gold do not face. This critical trade-off between pH-responsive toxicity and systemic stability is a central theme in ZnO NP research [22, 23].

Apart from their direct anticancer properties, ZnO-NPs contribute significantly to the modulation of the tumor microenvironment (TME). Through modulation of immune responses, angiogenesis, and tumor-associated signaling pathways, ZnO-NPs can disrupt the supportive ecosystem that tumors rely on for growth and metastasis [24, 25]. This multi-faceted action positions ZnO-NPs not merely as passive drug carriers but as active agents in the fight against cancer.

Despite these promising attributes, the clinical translation of ZnO-NPs is still in its infancy. Several critical questions remain regarding their long-term biocompatibility, potential toxicity, pharmacokinetics, and clearance from the body. Moreover, the synthesis methods, particle size, shape, surface charge, and coating materials can profoundly influence their biological interactions and therapeutic outcomes [26–28]. A systematic understanding of these parameters is essential for optimizing ZnO-NP formulations for clinical use.

Given the burgeoning interest in ZnO-NPs and the growing body of preclinical evidence supporting their antitumor potential, a comprehensive narrative review is warranted to synthesize current findings, highlight existing challenges, and outline future directions. Such a review is particularly valuable in the context of lung cancer, where the demand for more effective and less toxic therapies remains critical. By collating and critically analyzing the available literature, researchers and clinicians can gain deeper insights into the therapeutic mechanisms of ZnO-NPs, the superiorities they present over standard therapeutic approaches, and the technical aspects that must be addressed to ensure successful clinical adoption.

In this review, we aim to provide an in-depth overview of zinc oxide nanoparticles as a novel therapeutic approach against lung cancer. We will explore their physicochemical properties, mechanisms of cytotoxicity, strategies for tumor targeting, and current challenges in clinical translation. This narrative review serves to critically synthesize the molecular mechanisms of ZnO NP cytotoxicity and evaluate the current preclinical evidence, underscoring the necessity for further mechanistic and translational research in this promising domain.

Physical and chemical properties of ZnO nanoparticles for cancer therapy

With their unique physicochemical properties, zinc oxide nanoparticles are considered highly suitable for cancer therapy. These nanoparticles exhibit diverse morphologies including spherical, flower-like, and rod-shaped structures, with sizes typically ranging from 20–100 nm. The physicochemical properties of ZnO NPs, including particle size, surface area, and zeta potential, significantly influence their biological activity and therapeutic efficacy against cancer cells [29]. Several fabrication methods have been designed to generate ZnO NPs with tailored characteristics, with green synthesis methods gaining particular attention due to their eco-friendly nature and biocompatibility. As an example, Fusarium oxysporum fungal mycelia have been harnessed for the green synthesis of zinc oxide-superparamagnetic iron oxide-silver nanocomposites, designed to function as drug delivery systems for anticancer therapies [30]. Similarly, Portulaca oleracea has been employed in the eco-friendly synthesis of ZnO NPs, capitalizing on the plant's bioactive compounds to enhance the therapeutic potential of the resulting nanoparticles [31].

Zinc oxide can adopt various structural forms, including one-dimensional, two-dimensional, and three-dimensional architectures. This morphological diversity is not merely a structural catalog; it is a critical determinant of the nanoparticle's therapeutic action, as this linkage between morphology and therapeutic function is key to rational nanoparticle design [32]. For instance, one-dimensional (1D) structures like nanorods possess a high aspect ratio, which has been shown to significantly enhance cellular uptake and internalization compared to spherical particles [33, 34]. In contrast, complex three-dimensional (3D) structures, such as nanoflowers or urchin-like shapes, are highly valued for their extremely large, accessible surface-area-to-volume ratio. This increased surface area directly correlates with a higher catalytic capacity for generating reactive oxygen species (ROS), often leading to more potent anticancer effects [16, 35]. Two-dimensional (2D) structures like nanosheets, meanwhile, offer a different advantage, providing a large, flat plane ideal for high-capacity drug loading and functionalization. Therefore, the choice of morphology is a key a priori strategy in designing ZnO NPs for a specific therapeutic goal, such as maximizing direct cytotoxicity (via 3D nanoflowers) or optimizing drug delivery (via 2D nanosheets).

Zinc oxide exists in two primary crystallographic structures: hexagonal wurtzite and cubic zincblende. The wurtzite phase is the predominant form and remains thermodynamically stable at ambient conditions. ZnO crystallizes in a wurtzite (B4-type) structure at room temperature and atmospheric pressure. This hexagonal lattice, belonging to the P63mc space group, features two interwoven sublattices of Zn²⁺ and O²⁻ ions. Each Zn ion is surrounded by tetrahedra of O²⁻ ions, with the reverse occurring for each O²⁻ ion, creating a reciprocal arrangement. This arrangement of tetrahedral coordination leads to the development of polar symmetry along the hexagonal direction [36]. This inherent polarity governs many of ZnO's characteristics, including piezoelectric behavior and spontaneous polarization, and is a critical determinant in processes such as crystal growth, etching, and defect formation. Wurtzite ZnO exhibits four common face terminations: the polar (0001) face, terminated by Zn, and the (000.1) face, terminated by O, both oriented along the c-axis; along with the non-polar (11.20) (a-axis) and (10.10) faces, where Zn and O atoms are present in equal proportions. The polar faces exhibit unique physical and chemical traits, where the 'O'-terminated face reveals a notably different electronic structure in contrast to the remaining three 'O'-terminated faces [37].

The physical properties of ZnO nanoparticles are remarkable. When semiconductor materials are reduced to the nanometer scale, their physical properties alter due to "quantum size effects." Specifically, quantum confinement causes an increase in the band gap energy of quasi-one-dimensional (Q1D) ZnO, as confirmed by photoluminescence analyses [38].

The analysis of synthesized ZnO nanoparticles is commonly conducted using a variety of analytical methods, such as high-resolution transmission electron microscopy (HRTEM), scanning electron microscopy (SEM), X-ray diffraction (XRD), UV–visible spectroscopy, and FTIR (Fourier-transform infrared spectroscopy). The use of these methods verifies the crystalline structure, morphology, size distribution, and surface features of the nanoparticles. The size and shape of ZnO NPs significantly impact their cellular uptake and subsequent biological effects, with smaller particles generally demonstrating enhanced penetration into cancer cells and greater therapeutic efficacy [29].

Common synthesis methods and their therapeutic impact

The therapeutic efficacy of ZnO NPs is critically dependent on their synthesis method, as this dictates their size, morphology, surface defects, and purity.

• Sol–gel method: This is a common, low-temperature "wet chemistry" technique that allows for fine control over particle size and doping. By controlling the hydrolysis and condensation of zinc precursors, it can produce highly uniform, crystalline nanoparticles.

• Hydrothermal method: This method uses high-temperature and high-pressure water as a solvent. It is highly effective for synthesizing specific morphologies, such as the 1D nanorods discussed earlier, which are known to enhance cellular uptake.

• Green synthesis: As mentioned in Table 1, this "biosynthesis" approach uses natural extracts (from plants, fungi, or bacteria) as reducing and capping agents. This is a cost-effective and eco-friendly method that often results in highly biocompatible NPs with unique surface properties that can enhance their anticancer effect.

Table 1.

Summary of in vitro studies on ZnO NPs in lung cancer

Cell line	ZnO NP characteristics (size, shape, surface modification)	Concentration (µg/mL)	Key findings	References
A549 (NSCLC)	~ 20 nm, spherical, drug-loaded (Cisplatin/Gemcitabine)	Various	Significantly decreased cell viability, enhanced apoptosis, increased ROS, decreased mitochondrial membrane potential	[39]
A549 (NSCLC)	Fungus-synthesized, conjugated with Doxorubicin	IC50 = 0.34 (DOX-ZnO NPs)	Strong inhibition of proliferation, increased ROS, reduced mitochondrial membrane potential, enhanced apoptosis	[40]
A549 (NSCLC)	Methotrexate-conjugated	Various	Enhanced cytotoxic effect, induced apoptosis, activated caspase-9, -8, and -3	[41]
A549 (NSCLC)	Green synthesized using Neem extract	100–250	Significant decrease in cell viability, IC50 = 138.50 after 48 h	[34]
Small-cell lung cancer cells (N417, H82, H187)	~ 20 nm	Various	Genotoxic, low viability, induced ROS and DNA leakage	[11]
A549 (NSCLC)	ZnO-CMC-MGA nanocomposites	Various	Enhanced cytotoxicity compared to MGA alone	[42]
A549 (NSCLC)	45–60 nm; Nearly spherical and hexagonal quartzite shape; Synthesized from Mangifera indica (mango) leaves	25 µg/mL (for cytotoxicity); variable (for antioxidant assay)	Significant cytotoxicity at 25 µg/mL comparable to cyclophosphamide; Antioxidant activity increased with concentration; ZnO NPs stable and eco-friendly	[43]
SKLC-6	Gd-doped; Grain size 9 nm; High purity and crystalline; Synthesized via chemical precipitation	10 and 20 µg/mL	Dose-dependent radiosensitivity enhancement with SER of 1.47 and 1.61; Induced apoptosis, G1 arrest, and micronucleus formation; Downregulation of XRCC2/XRCC4 genes; Enhanced CT and MR imaging contrast	[44]

The choice of method is not trivial. For example, methods that result in a higher density of surface defects (like oxygen vacancies) can lead to a significant increase in ROS generation, enhancing the nanoparticle's inherent cytotoxicity [45]. Conversely, methods that produce highly stable, uniform particles may be better suited for drug delivery applications [46].

Anticancer properties of zinc oxide nanoparticles in lung cancer

It is critical to distinguish between two primary therapeutic approaches reviewed in this manuscript, which the reviewer has correctly identified. The first approach utilizes the inherent anticancer activity of bare (unfunctionalized) ZnO NPs. The second approach uses ZnO NPs as a nanocarrier system to be coated or encapsulated with other chemotherapeutic drugs (e.g., cisplatin, doxorubicin).

To create a clear and logical narrative, the following "Mechanisms of Action" section will focus primarily on the inherent cytotoxic properties of the ZnO nanoparticles themselves (e.g., ROS generation, pH-driven Zn²⁺ ion release). After establishing these baseline mechanisms, the subsequent "Zinc Oxide Nanoparticles as a Drug Delivery System" section will then explore the synergistic advantages of using ZnO NPs as a platform for co-delivering established drugs.

Several in vitro investigations have examined the anticancer potential of ZnO NPs in the treatment of lung cancerConsistent findings across these studies highlight the cytotoxic activity of ZnO nanoparticles (NPs) against different lung cancer cell lines, including A549 (non-small cell lung adenocarcinoma), CAL 27 (a head and neck squamous cell carcinoma line often used as a model), and small-cell lung cancer cells [47]. Cell viability assays, such as MTT assays, have shown a dose-dependent reduction in lung cancer cell survival upon exposure to ZnO NPs, with some studies reporting IC50 values (the concentration required to inhibit 50% of cell growth) [48–50]. Notably, several investigations have indicated that ZnO NPs exhibit selective toxicity, demonstrating higher cytotoxicity towards cancer cells compared to normal lung-derived cells and other normal cell types [11].

The anticancer activity of ZnO NPs is primarily driven by the induction of reactive ROS in cancer cells. The overproduction of ROS creates oxidative stress, leading to damage to critical cellular components, including DNA, proteins, and lipids, and eventually initiating apoptotic or necrotic pathways. Furthermore, studies have shown that ZnO NPs can directly interact with DNA, leading to DNA damage and leakage from the nuclei of lung cancer cells [47].

In vivo preclinical models of lung cancer have also been used to evaluate the therapeutic efficacy of ZnO NPs. Studies employing orthotopic mouse models, where human small-cell lung cancer cells are engrafted into the lungs of mice, have yielded promising outcomes. Intravenous administration of ZnO NPs in these models resulted in reduced viability of the cancer cells and genotoxic effects within the tumors, without causing observable adverse effects in the normal lung tissues or other organs [11]. Moreover, researchers have explored the potential of combining ZnO NPs with conventional chemotherapy drugs to enhance their anticancer effects. For instance, studies have demonstrated that loading cisplatin and gemcitabine onto ZnO NPs can significantly improve their cytotoxic action against non-small cell lung cancer cells in xenograft models, leading to enhanced inhibition of tumor formation [39].

Mechanisms of action

ZnO NPs exhibit anticancer activity in lung cancer cells through several interconnected mechanisms, with the production of reactive ROS being a pivotal component [51]. ZnO NPs, functioning as semiconductors, can promote the production of ROS, such as superoxide radicals, hydrogen peroxide, and hydroxyl radicals, upon interacting with cellular components, especially under specific conditions like UV radiation exposure [11, 52, 53]. This surge in ROS levels overwhelms the antioxidant defense mechanisms of cancer cells, leading to oxidative damage to crucial biomolecules, including DNA, lipids, and proteins [54].

Overwhelming the oxidative stress threshold in lung cancer

The primary mechanism of ZnO NP cytotoxicity is the induction of overwhelming oxidative stress [55]. This strategy is uniquely effective against lung cancer cells, which often exhibit a flawed redox balance as part of their pathogenesis. Due to rapid proliferation and oncogenic signaling, many cancer cells exist in a state of high basal oxidative stress, operating near their maximum antioxidant capacity [56, 57].

ZnO NPs, functioning as semiconductors, catalyze the production of reactive oxygen species (ROS), including superoxide radicals, hydrogen peroxide, and hydroxyl radicals [12–14, 58, 59]. This sudden influx of ROS effectively "pushes" the pre-stressed cancer cells over a toxic threshold, a state that healthy cells with lower basal ROS levels can manage. This leads to catastrophic oxidative damage to crucial biomolecules, including lipids, proteins, and DNA [33, 34]. This catastrophic oxidative damage to biomolecules is not just random; it directly modulates key oncogenic signaling pathways. High ROS levels are known to suppress the pro-survival PI3K/Akt pathway and activate the stress-response MAPK/ERK pathway, which can, in turn, trigger apoptosis [60–63]. Therefore, the ROS generated by ZnO NPs acts as a signaling molecule that shuts down the cancer cell's "grow and survive" signals.

The oxidative stress induced by ZnO NPs frequently triggers apoptosis, a programmed cell death pathway essential for eliminating damaged or unwanted cells. Studies indicate that ZnO NPs activate caspase cascades, a series of enzymes involved in apoptosis, and promote the upregulation of pro-apoptotic proteins like Bax, alongside the downregulation of anti-apoptotic proteins such as Bcl-2 [58, 59]. ZnO NPs also have the ability to disturb the mitochondrial membrane potential, a key element in cellular energy production, which triggers the release of pro-apoptotic factors and activates cell death mechanisms [52, 54, 58, 59].

Beyond inducing apoptosis, ZnO NPs also suppress mitotic progression by enforcing cell cycle arrest. This is not merely a side effect but a distinct mechanistic action, as demonstrated in a case study by Rani et al. [64] on human lung cancer (A549) cells. Using flow cytometry analysis, the authors provided a clear example of this mechanism, showing that ZnO NP exposure caused a significant, dose-dependent accumulation of cells in the G2/M phase. This blockade, which prevents cells with damaged DNA from proceeding through mitosis and replicating, was linked to the nanoparticles' interference with the microtubule spindle apparatus and the modulation of key cell cycle regulators like cyclin B1 and Cdk1. This mechanism effectively halts tumor growth at its source: cellular replication.

Exploiting the acidic tumor microenvironment

The TME in lung cancer, as in most solid tumors, is characterized by extracellular acidosis due to the Warburg effect. This pathogenic feature creates a unique vulnerability that ZnO NPs can exploit. ZnO NPs possess pH-sensitive solubility, meaning they are relatively stable at the physiological pH of healthy tissue (7.4) but dissolve rapidly in acidic environments.

This pH-responsive dissolution is a key component of their selective action. As NPs are endocytosed, they are exposed to the highly acidic environment of the endosome (pH ~ 5.5), where they break down and release a high concentration of cytotoxic Zn²⁺ ions directly inside the cell. This "Trojan Horse" mechanism, triggered by a key feature of the cancer cell's own pathogenesis, enhances targeted toxicity while sparing healthy tissues [17, 41]. This flood of free Zn²⁺ ions is, in itself, a primary cytotoxic mechanism separate from ROS generation. High intracellular zinc levels are profoundly toxic, as the excess ions can disrupt cellular homeostasis by outcompeting other cations (like Mg²⁺ and Ca²⁺) for enzymatic binding sites. This ion-mediated toxicity is reported to inhibit mitochondrial respiratory chain enzymes and interfere with calcium signaling, thereby initiating a parallel, ROS-independent pathway for apoptosis [65, 66]. This dual mechanism of action—simultaneous ROS generation and ion-mediated toxicity—is what makes ZnO NPs a particularly robust anticancer agent [65, 67, 68].

Restoring apoptotic signaling

A hallmark of lung cancer pathogenesis is the acquisition of resistance to apoptosis, often through the mutation of tumor suppressor genes like TP53 or the dysregulation of the Bcl-2 protein family [69]. ZnO NPs have been shown to directly counter this resistance by activating intrinsic apoptotic pathways.

The oxidative stress induced by ZnO NPs triggers mitochondrial membrane potential disruption, a key event that cancer cells try to suppress [33, 34, 58, 59]. This disruption forces the release of pro-apoptotic factors, activating the caspase cascade. Studies consistently show that ZnO NP exposure leads to the upregulation of the pro-apoptotic protein Bax and the downregulation of the anti-apoptotic protein Bcl-2, effectively re-engaging the apoptotic "suicide" program that the lung cancer cell had silenced [33, 34, 70].

Figure 2 provides a schematic overview of these interconnected pathways, highlighting how ZnO NP exposure leads to three primary outcomes for the lung cancer cell: ROS-induced oxidative stress, activation of the apoptotic pathway (via Bax/Bcl-2 modulation), and a G2/M cell cycle blockade.

Fig. 2.

Mechanisms of anticancer activity of zinc oxide nanoparticles (ZnO NPs) in lung cancer cells. This figure highlights the various pathways through which ZnO nanoparticles induce cytotoxicity in lung cancer cells. Exposure to ZnO nanoparticles results in the formation of various reactive oxygen species, including superoxide, H₂O₂, and hydroxyl radicals, triggering oxidative stress that compromises the integrity of DNA and other critical cellular components. Oxidative stress initiates apoptotic signaling through the upregulation of Bax, suppression of Bcl-2 expression, and subsequent activation of caspase-3. Mitochondrial membrane disruption further promotes apoptosis. Furthermore, ZnO nanoparticles disrupt cell cycle progression at the G2/M checkpoint, effectively suppressing the growth of cancer cells

A critical analysis of the preclinical data, both in vitro and in vivo, reveals significant experimental inconsistencies that are a major hurdle for the field. As Tables 1 and 2 demonstrate, there is a profound lack of standardization that makes direct comparison between studies nearly impossible. This includes wide variation in synthesis methods (e.g., green synthesis, chemical precipitation, doping), fundamental differences in physicochemical properties (particle size, shape, surface modifications), and a failure to adopt standardized dosing metrics. Studies report concentrations in µg/mL or mg/kg, which are not comparable without knowing the precise nanoparticle characteristics and hydrodynamic size. This experimental heterogeneity directly impacts reproducibility and is a fundamental barrier to establishing a clear dose–response relationship, making it difficult to determine which formulations are truly the most promising for clinical translation.

Table 2.

Summary of in vivo studies on ZnO NPs in lung cancer models

Animal model	Type of lung cancer model	ZnO NP characteristics, dose, and schedule	Key findings	References
Mice (BALB/c nude)	Orthotopic xenograft(N417 Small-cell lung cancer)	NP Type: Bare ZnO NPs Size: ~ 20 nm Dose/Route: 0.25 mg/kg, Intravenous (IV) Schedule: 3 total injections (Days 15, 22, and 29 post-tumor injection)	Inhibited tumor proliferation; Showed in vivo genotoxicity (TUNEL staining); NPs accumulated in tumors; Low toxicity to normal lung cells; No adverse effects or weight loss observed	[11]
Mice (BALB/c nude)	Xenograft (Subcutaneous)(A549 NSCLC)	NP Type: Loaded with Cisplatin (Cp) & Gemcitabine (Gem) Size: ~ 20 nm Dose/Route: Dose not specified, Intraperitoneal (IP) Schedule: Once every other day	Significantly enhanced inhibition of tumor formation compared to cisplatin and gemcitabine administered alone or as a simple (Cp + Gem) combination	[39]
Mice (Swiss albino)	Benzo(a)pyrene (BAP)-induced lung cancer	NP Type: Loaded with Syringic Acid (ZnO-SYR) Size: ~ 120 nm Dose/Route: 20 mg/kg b.wt., Oral Schedule: Not specified (Administered from week 12 to 18)	Restored body weight and attenuated serum marker enzymes (AHH, GGT, LDH); Ameliorated cytokine levels (TNF-α, IL-6);	[47]

With this critical caveat in mind, in vitro studies, as summarized in Table 1, have still consistently demonstrated that ZnO NPs, regardless of their specific synthesis, reliably induce cytotoxicity in lung cancer cell lines (predominantly A549) through a common mechanistic core: the induction of oxidative stress (seen as increased ROS), disruption of mitochondrial membrane potential, and subsequent activation of apoptosis. An overarching conclusion is that functionalization is a highly effective strategy to boost efficacy. The studies highlight two particularly promising functionalization strategies: 1) The conjugation of ZnO NPs with conventional chemotherapeutics like Doxorubicin [43] and Methotrexate [44], which creates a synergistic system that enhances the cytotoxic effect, and 2) The use of "green synthesis" methods using plant extracts [16, 71–73], which are shown to be a viable, eco-friendly route to producing effective nanoparticles.

Potential for anti-angiogenic effects

The pathogenesis of advanced lung cancer relies on angiogenesis, the formation of new blood vessels, which is heavily promoted by factors such as Vascular Endothelial Growth Factor (VEGF) [74]. While the primary focus has been on cytotoxicity, the nanoparticles' role in modulating the TME, as noted in the introduction, suggests a potential anti-angiogenic mechanism [24–28]. This remains a critical area for future mechanistic investigation, as a therapy that simultaneously kills cancer cells and chokes off their blood supply would be a significant advancement over conventional treatments.

Unpacking the "selectivity" of ZnO nanoparticles: a mechanistic view

A central promise of the title is the "selective" action of ZnO NPs against cancer cells, and this claim is a primary driver of research. However, this "selectivity" is not a single property but an emergent phenomenon resulting from several, simultaneous mechanistic interactions. While passive targeting via the Enhanced Permeation and Retention (EPR) effect is often cited, this mechanism is not specific to ZnO NPs and has limitations. The true selectivity is more active and is rooted in exploiting the distinct pathological vulnerabilities of lung cancer cells.

1. pH-driven selectivity (the "trojan horse"): As discussed previously, the acidic tumor microenvironment (TME) and, more importantly, the highly acidic endosomes (pH ~ 5.5) of rapidly internalizing cancer cells are the primary drivers of selectivity. ZnO NPs are uniquely pH-sensitive. They dissolve rapidly in these acidic compartments, releasing a flood of cytotoxic Zn²⁺ ions inside the cancer cell. In contrast, healthy cells at physiological pH (7.4) do not trigger this rapid dissolution, leaving the NPs comparatively inert and sparing the tissue from toxicity [20, 55].

2. Redox-based selectivity (pushing cells "over the edge"): Lung cancer cells, due to their oncogenic signaling and high metabolic rate, already exist in a state of high "basal" oxidative stress. Their antioxidant defense systems (like glutathione) are operating near maximum capacity to manage this [20, 75]. The ROS generated by ZnO NPs is, therefore, a "tipping point"—it's an oxidative load that a pre-stressed cancer cell cannot handle, pushing it into apoptosis. Healthy cells, with their low basal ROS and full antioxidant capacity, can easily neutralize the same dose of ROS, resulting in minimal toxicity.

Therefore, the selectivity of ZnO NPs is a robust, multi-faceted mechanism. It is a scientific and "smart" system that exploits the cancer's own pathogenic properties (acidosis and high ROS) to preferentially kill it.

3. Electrostatic selectivity (surface charge): The claim of "cationic nature" must be clarified scientifically. ZnO NPs have a high isoelectric point (pI), typically around pH 9–10. This means that in physiological environments (pH ~ 7.4), which is below the pI, the nanoparticle surface becomes protonated and exhibits a stable positive surface charge. Cancer cell membranes are known to be net negative (anionic) due to an abundance of sialic acid and phosphatidylserine. This creates a strong electrostatic attraction, or "affinity," that promotes the preferential binding and subsequent endocytosis of the positively-charged ZnO NPs by cancer cells over healthy cells, enhancing their selective cytotoxic potential [75, 76].

4. Exploiting core molecular vulnerabilities: The selectivity of ZnO NPs also stems from their ability to target fundamental cancer-driving pathways. For example, many lung cancers feature p53 mutations, which cripples the cell's ability to repair DNA. This makes them hyper-sensitive to the DNA damage induced by ZnO NP-generated ROS [61, 77]. In fact, studies on lung cancer cells (A549) confirm that ZnO NPs trigger apoptosis specifically through a ROS-generated p53/Bcl-2/Bax signaling pathway [78]. Furthermore, the high levels of ROS are known to modulate key pathways like the HIF-1α/VEGF axis, which is critical for VEGF-driven angiogenesis and tumor survival [63]. By generating ROS, ZnO NPs can thus simultaneously induce DNA damage in cells that can't repair it and disrupt the signaling that builds the tumor's blood supply.

Zinc oxide nanoparticles as a drug delivery system

Beyond their direct anticancer activity, ZnO NPs have also been explored as a promising drug delivery system for lung cancer therapy [16, 73]. Several chemotherapeutic drugs, including cisplatin, gemcitabine, doxorubicin, and methotrexate, have been successfully loaded onto ZnO NPs to enhance their delivery to lung cancer cells [39, 73, 79]. Various strategies, such as physical adsorption, encapsulation within the nanoparticle matrix, and chemical conjugation to the nanoparticle surface, have been employed for drug loading [16, 35].

To improve the specificity of drug delivery, researchers have functionalized the surface of ZnO NPs with targeting ligands, such as antibodies, peptides, and folic acid. These ligands can recognize and bind to receptors that are overexpressed on the surface of lung cancer cells, facilitating targeted uptake of the drug-loaded nanoparticles [16, 35].

Perhaps the most powerful strategy for tumor-selective delivery is exploiting the inherent pH-responsive dissolution of ZnO NPs. The nanoparticle is designed to act as a stable 'gatekeeper,' keeping its chemotherapeutic payload (such as doxorubicin or methotrexate) safely encapsulated while circulating in the bloodstream at physiological pH (7.4). However, upon accumulation in the tumor and subsequent endocytosis by cancer cells, the nanoparticle is exposed to the highly acidic environment of the endosome (pH ~ 5.5). This acidic trigger causes the ZnO NP matrix to rapidly dissolve, or 'break down,' releasing its high-concentration drug payload directly inside the cancer cell. This "Trojan Horse" mechanism is a key advantage of ZnO NPs, as it enhances the cytotoxic effect specifically at the tumor site while minimizing the off-target systemic toxicity associated with the free drug [73].

Studies have demonstrated that drug-loaded ZnO NPs often exhibit enhanced anticancer activity compared to the free drug in lung cancer models. This improved efficacy is attributed to increased drug concentration at the tumor site, enhanced cellular uptake, and controlled drug release.

Furthermore, a significant advantage of using ZnO NPs as a co-delivery platform is their ability to actively overcome multidrug resistance (MDR), a primary cause of chemotherapy failure. Many lung cancer cells overexpress the P-glycoprotein (P-gp) efflux pump, an ATP-dependent protein that actively ejects chemotherapy drugs from the cell. The massive ROS generation induced by ZnO NPs, however, causes severe mitochondrial damage and a subsequent depletion of intracellular ATP. This effectively 'shuts down' the P-gp pump by starving it of energy, thereby trapping the co-delivered drug (like doxorubicin) inside the cancer cell, restoring its therapeutic efficacy [80, 81].

Comparison with existing lung cancer therapies

When comparing ZnO NPs to conventional therapies, a direct quantitative comparison is complex because ZnO NPs are a heterogeneous class of materials, not a single drug. Standard therapies like cisplatin have a defined IC50, whereas the IC50 of ZnO NPs can vary by orders of magnitude depending on their size, synthesis, and cell line used (as seen in Table 1).

The primary advantage of ZnO NPs is not in their potency (cisplatin is often more potent at a lower molar concentration) but in their synergy and selectivity. As comparative data from Hu et al. [36] demonstrated, the combination of ZnO-NPs(Cp/Gem) led to significantly greater tumor inhibition in xenograft models than the free Cp + Gem combination alone. This suggests the main role of ZnO NPs is as a delivery system that enhances the efficacy of existing drugs, in part by overcoming P-glycoprotein-based multidrug resistance.

However, this comparison also highlights their main drawback: pharmacokinetics (PK) and biodistribution. Small-molecule drugs like cisplatin have predictable PK. In contrast, nanoparticles are rapidly identified as foreign and cleared by the reticuloendothelial system (RES). This leads to high, unintended accumulation in the liver and spleen and a short circulating half-life, which are major disadvantages that must be overcome with stealth coatings (e.g., PEGylation) to improve tumor targeting and delivery.

Efficacy in preclinical models

The potential of ZnO NPs as an anticancer agent for lung cancer has been extensively investigated in preclinical models, both in vitro and in vivo.

In vitro studies, as summarized in Table 1, have consistently demonstrated the ability of ZnO NPs to induce cytotoxicity, apoptosis, and ROS production in various lung cancer cell lines [47]. These effects exhibit a dose-dependent relationship, with higher levels of ZnO nanoparticles inducing greater levels of cell death. Notably, some studies have shown that ZnO NPs exhibit selectivity, being more toxic to cancer cells than to normal lung-derived cells [11]. Furthermore, the combination of ZnO NPs with conventional chemotherapeutic agents has shown synergistic effects, enhancing the overall anticancer activity [79].

The in vivo data in Table 2 translates these in vitro findings into preclinical models, providing two critical overarching conclusions. First, ZnO NPs demonstrate significant tumor inhibition in various mouse xenograft models, confirming their efficacy beyond a petri dish [11–14, 52, 53]. Second, and perhaps most critically for their "bench to bedside" potential, they appear to exhibit a high degree of selective toxicity. This is a consistent trend across the studies, with Tanino et al. [11] noting low toxicity to normal lung cells and Yang et al. [55] observing that their formulation restored body weight and ameliorated cytokine levels. The most promising strategy highlighted by these studies is the use of ZnO NPs as a synergistic combination platform. Loading the NPs with cisplatin/gemcitabbine [39] or syringic acid [55] resulted in significantly enhanced tumor inhibition, suggesting that the primary in vivo utility of ZnO NPs may be as a targeted, toxicity-reducing drug delivery system.

Fundamental hurdles and future research directions

The translation of promising preclinical findings on ZnO NPs into effective clinical therapies for lung cancer faces several challenges and limitations. One significant hurdle lies in the consistent and scalable synthesis of ZnO NPs with precisely controlled size, shape, and surface properties. Ensuring the quality and reproducibility of nanoparticle production is crucial for clinical applications, necessitating robust characterization techniques [16, 82–84].

Beyond active targeting, surface functionalization is also critical for improving in vivo pharmacokinetics by evading immune detection. Bare nanoparticles are rapidly recognized by plasma proteins (opsonins), leading to opsonization and subsequent rapid clearance by the reticuloendothelial system (RES), primarily in the liver and spleen [85]. To overcome this, "stealth coatings" are applied. The most common strategy is PEGylation (grafting polyethylene glycol) to the NP surface, which creates a neutral, hydrophilic layer that sterically hinders protein binding [86]. Other natural polymers, such as chitosan and albumin, are also used to create biomimetic "stealth" surfaces [86, 87]. These coatings effectively "cloak" the nanoparticle, significantly reducing RES uptake, prolonging blood circulation half-life, and ultimately increasing the probability of tumor accumulation.

A major hurdle for in vivo efficacy is the complex pharmacokinetics and biodistribution of ZnO NPs. After intravenous injection, bare nanoparticles are rapidly opsonized (coated by plasma proteins), leading to recognition and rapid uptake by the reticuloendothelial system (RES). This results in significant, undesirable nanoparticle accumulation in RES organs, primarily the liver (Kupffer cells) and spleen, as well as the lungs [88–90]. This not only reduces the dose available to the tumor but also poses a risk of organ-specific toxicity. Furthermore, the clearance pathways for ZnO NPs are complex and size-dependent. While very small NPs (< 5.5 nm) can be cleared renally (via urine), most therapeutic ZnO NPs are larger and rely on slower hepatobiliary clearance (via the liver and feces) [91]. However, the unique challenge for ZnO NPs is their dissolution in vivo. This means clearance is a dual process: the body must clear both the intact nanoparticles and the free Zn²⁺ ions released by premature dissolution. A full understanding of this biodistribution, dissolution, and clearance profile is currently lacking and is essential to confirm long-term biocompatibility and establish a safe therapeutic window [92].

Currently, there is a lack of extensive clinical trial data on the use of ZnO NPs for lung cancer treatment. Navigating the regulatory pathways for nanomedicines can be complex, and standardized protocols for preclinical and clinical evaluation are needed to facilitate their translation into approved therapies.

Future research should focus on addressing the current limitations and further exploring the potential of ZnO NPs in lung cancer treatment. Combination therapies involving ZnO NPs and other anticancer modalities, such as radiotherapy, immunotherapy, and targeted therapies, hold promise for achieving synergistic effects and improving treatment outcomes [16, 93, 94]. Developing novel drug delivery systems that combine ZnO NPs with other nanomaterials or strategies could also enhance their efficacy. Developing novel combination nanoplatforms for hybrid delivery approaches could also enhance efficacy. For example, encapsulating ZnO NPs within a larger liposome or polymeric nanoparticle could leverage the stability and stealth properties of the outer carrier while still exploiting the pH-responsive, cytotoxic payload of the inner ZnO NP core. This is particularly relevant for developing sophisticated co-delivery systems that combine chemotherapeutics with emerging RNA-based therapies. Given their positive surface charge at physiological pH, ZnO NPs are promising non-viral vectors for carrying anionic payloads like siRNA or miRNA. This hybrid approach could be used to simultaneously deliver a cytotoxic drug while silencing critical oncogenes (e.g., KRAS) or non-coding RNAs that drive multidrug resistance, offering a powerful, multi-pronged attack on lung cancer [95].

Moreover, future mechanistic studies must investigate the interplay between ZnO NPs and other critical TME factors. For instance, the impact of hypoxia (low oxygen) on ZnO NP dissolution and ROS generation is poorly understood. Similarly, the potential immunomodulatory effects of ZnO NPs—whether they promote or suppress an anti-tumor immune response—remain a critical, unexplored area for clinical translation.

Further refinement of targeted delivery systems through sophisticated surface engineering strategies is needed to achieve highly specific targeting of different subtypes of lung cancer cells. Exploring alternative administration routes, such as inhalation or direct intratumoral injection, could improve local drug concentration within the lungs [96].

The potential of ZnO NPs for theranostic applications, combining diagnosis and therapy, warrants further investigation. Developing ZnO NP-based sensors for early detection of lung cancer and real-time monitoring of treatment response could revolutionize disease management [16, 84, 97].

In-depth preclinical investigations are vital to determine the prolonged safety profile and potential unintended effects of ZnO nanoparticles. Well-structured and controlled clinical studies are crucial to assess both the effectiveness and safety profile of ZnO nanoparticles in patients with lung cancer. As summarized in Fig. 3, these key hurdles span the entire translational pipeline, from consistent nanoparticle synthesis and targeted delivery to the lack of long-term safety and clinical trial data (Fig. 3).

Fig. 3.

ZnO NPs clinical translation challenges

Conclusion

Zinc oxide nanoparticles represent a highly promising, multi-functional platform for lung cancer therapy. This review of the preclinical evidence has moved beyond a general summary to reveal that their efficacy is rooted in a sophisticated, multi-pronged mechanism. The central promise of ZnO NPs lies in their "smart" selectivity, which is not passive, but an active exploitation of the unique lung cancer pathophysiology.

We have identified three core mechanistic pillars of this selectivity: 1) pH-Driven Selectivity, where the acidic tumor microenvironment triggers nanoparticle dissolution and a "Trojan Horse" release of toxic Zn²⁺ ions; 2) Redox-Based Selectivity, where ZnO NP-generated ROS pushes the already high-stress cancer cells "over the edge" into apoptosis, while sparing healthy cells; and 3) Electrostatic Selectivity, where the positive surface charge of ZnO NPs (at physiological pH) promotes their binding to the anionic membranes of cancer cells.

Beyond their potent inherent cytotoxicity, ZnO NPs are also a powerful drug delivery system. They can be loaded with standard chemotherapeutics, and their ability to deplete intracellular ATP provides an additional, powerful mechanism for overcoming multidrug resistance by inhibiting P-glycoprotein efflux pumps.

Despite this promise, the field is at a critical preclinical juncture. We have identified significant hurdles, including a lack of standardization in synthesis and dosing, and a poor understanding of in vivo pharmacokinetics, biodistribution, and RES clearance. Future research must focus on solving these fundamental challenges—for instance, by developing "stealth" coatings and hybrid nanoplatforms. Only then can we unlock the full therapeutic potential of ZnO nanoparticles and begin the critical work of translating these findings from the bench to the bedside.

Author contributions

Hamdi Nsairat, Waleed K. Abdulsahib, S. Renuka Jyothi, Priya Priyadarshini Nayak, Ashish Singh Chauhan, Siya Singla, Fadhil Faez Sead, Djamila Polatova involved in the conception, design, and drafting of the manuscript.

Funding

Not applicable.

Data availability

No datasets were generated or analysed during the current study.

Declarations

Ethics approval and consent to participate

Not applicable.

Consent for publication

Not applicable.

Competing interests

The authors declare no competing interests.