A Comprehensive Review on Copper Oxide Nanoparticles: Physicochemical Parameters, Pharmacological Potential and Pathophysiological Effects

Abstract

Copper oxide nanoparticles (CuONPs) have attracted considerable attention in biomedical research owing to their unique physicochemical properties, high surface reactivity, and versatile biological activities. Their nanoscale dimensions have enabled diverse applications in antimicrobial therapy, anticancer treatment, biosensing, and drug delivery. However, increasing biomedical and environmental exposure to CuONPs has raised significant concerns regarding their biodistribution, biocompatibility, and potential toxicological effects. Unlike previous reviews that have generally addressed these aspects separately or with limited integration, this review integrates the physicochemical characteristics, exposure routes, biodistribution, pharmacological activities, and toxicological effects of CuONPs to provide a comprehensive understanding of their biomedical potential and safety. Emphasis is placed on the influence of inhalational, oral, dermal, and parenteral exposure routes and biological interactions of CuONPs, including their cellular internalization, protein corona formation, systemic translocation, and organ-specific accumulation. Furthermore, the review comprehensively examines the therapeutic applications of CuONPs, highlighting their antibacterial, anticancer, antidiabetic, and antioxidant properties, while also discussing their associated toxicity such as oxidative stress, inflammatory responses, genotoxicity, and regulated cell death pathways such as apoptosis, autophagy, ferroptosis, and cuproptosis. Evidence from both in vitro and in vivo studies demonstrates that CuONPs can induce significant pulmonary, hepatic, renal, neurological, reproductive, and haematological toxicities depending on their physicochemical properties and exposure conditions. Overall, this review highlights the dual role of CuONPs as both promising nanotherapeutic agents and potential toxicological hazards, emphasizing the need for standardized biological evaluation, safer nanoparticle design strategies, and improved understanding of biodistribution and long-term biological effects to support their future clinical translation.

Introduction

Nanotechnology, derived from the Greek words’ “nanos” (very small) and “techne” (art or craft), refers to the manipulation of matter at the nanoscale (1–100 nm) to enhance physicochemical and biological properties.^1,2 The use of nanoparticles dates back to ancient Egypt, where galena (PbS) nanoparticles formed from lime, lead oxide, and water were used as black hair dye, representing an early example of nanoparticle synthesis.³ Modern nanotechnology originated from Richard Feynman’s 1959 lecture “There’s Plenty of Room at the Bottom”, in which he proposed atom-by-atom manipulation of matter, earning him the title “Father of Nanotechnology”.⁴ In 1974, Norio Taniguchi formally defined nanotechnology as material manipulation at the atomic and molecular level,⁵ while K. Eric Drexler later advanced the field through the concept of molecular nanotechnology in 1986.⁶ Further progress was achieved in 1991 with Sumio Iijima’s discovery of carbon nanotubes and Paul Anastas’ introduction of green chemistry to reduce the environmental impact of chemical synthesis.^7,8 A timeline highlighting the historical development and major milestones in nanotechnology is presented in Figure 1.

Figure 1.

Historical milestones in the development of nanotechnology and green nanoscience. The timeline illustrates key events that contributed to the evolution of nanotechnology, beginning with the use of naturally occurring lead sulfide (PbS, galena) nanoparticles as a black pigment in Ancient Egypt. In 1959, Richard P. Feynman proposed the concept of manipulating matter at the atomic scale in his lecture “There’s Plenty of Room at the Bottom”. In 1974, Norio Taniguchi formally introduced the term nanotechnology to describe material processing and manipulation at the nanometer scale. In 1986, K. Eric Drexler advanced the concept of molecular nanotechnology. In 1991, Sumio Iijima reported the discovery of carbon nanotubes, while Paul Anastas introduced the principles of green chemistry, laying the foundation for the development of sustainable nanomaterial synthesis strategies.

Nanoparticles are broadly classified into three categories: organic, inorganic, and carbon-based. Organic nanoparticles comprise nanoparticles made from proteins, carbohydrates, polymers, lipids or any other organic compounds, whereas inorganic nanoparticles include metals and metal oxides, and carbon-based nanoparticles are composed solely of elemental carbon atoms.⁹ In the biomedical field, nanoparticles have gained significant attention due to their wide-ranging applications, including disease diagnosis and imaging techniques such as magnetic resonance imaging (MRI), computed tomography, and ultrasound, as well as drug delivery systems designed to improve therapeutic efficacy.^10,11 Currently, gadolinium-based contrast agents are widely used in clinical MRI to enhance image contrast; however, their use is limited by concerns such as nephrotoxicity, brain deposition, and lack of tissue specificity.¹² In this regard, nanoparticle-based contrast agents offer promising alternatives owing to their improved biocompatibility, prolonged circulation time, and potential for functionalization with targeting ligands for imaging and therapy.¹² In addition, nanoparticles play an important role in tissue engineering,¹³ where they are utilized in applications such as deoxyribonucleic acid (DNA) transfection¹⁴ and gene delivery.¹⁵ Established applications include technetium-99m colloids for lymphatic imaging and sentinel lymph node detection, as well as gold and silver nanoparticles in rapid diagnostic tests.¹⁶

Despite several successful applications, the broader clinical adoption of nanoparticles remains limited. Although advanced systems such as surface-enhanced Raman scattering based nanoparticles demonstrate high diagnostic sensitivity, their clinical translation is hindered by issues such as poor reproducibility and measurement variability.¹⁶ The major obstacle faced in the successful clinical translation of the nanomedicines are due to the poor standardization, inadequate characterization, inconsistent study design, insufficient safety evaluation and regulatory challenges.¹⁷ The regulation of nanomedicines is complicated because of their inherent physicochemical characteristics, including pharmacokinetic (PK) and pharmacodynamic (PD) profiles that differ from those of the corresponding bulk materials and loaded therapeutic agents.¹⁸ These challenges are even more pronounced for copper-based nanoparticles, where limited and fragmented data on PK, PD, and long-term toxicity continue to restrict clinical validation.¹⁹ Moreover, a significant gap remains between promising preclinical findings and clinical application, largely due to limited clinical relevance, lack of long-term fate, and challenges associated with large-scale production and reproducibility.²⁰ Furthermore, human exposure to copper-containing particulates may occur in occupational settings such as mining, smelting, and copper refining, where workers can inhale copper dust and fumes.²¹ Workplace airborne concentrations of copper are routinely monitored using standardized methods developed by the National Institute for Occupational Safety and Health and the Occupational Safety and Health Administration in which air samples are collected on cellulose ester membrane filters and subsequently analysed for copper content.²² Although no standardized biomonitoring protocol has been established for assessing occupational copper exposure, urinary copper concentrations can be measured using inductively coupled plasma–mass spectrometry and have been applied in population-based surveys such as the National Health and Nutrition Examination Survey.²² However, Occupational Safety and Health Administration has established a permissible exposure limit of 0.1 mg/m³ for copper fumes as an 8 hours (h) time-weighted average, indicating that workers average exposure during a work shift should not exceed this concentration.²³

Among metal oxide nanoparticles, CuONPs have attracted considerable interest due to their extensive applications in biomedical, energy storage systems, nanofluids, sensors and in catalysis.^24–28 However, their potential benefits are accompanied by notable safety concerns. CuONPs are known to generate reactive oxygen species (ROS), leading to oxidative stress and subsequent damage to cellular components.²⁹ They can also trigger the release of pro-inflammatory cytokines³⁰ and induce toxicity in various organs, including the liver,³¹ kidneys,³² spleen,³³ and lungs.³⁴ Furthermore, CuONPs may disrupt immune function and cause DNA damage, contributing to broader biological complications.^35,36 Compared with other metallic nanoparticles such as gold and silver nanoparticles, whose synthesis is often associated with high production costs and complex fabrication procedures,^37–40 copper-based nanoparticles are particularly attractive because of the high natural abundance, low cost, and easy availability of copper precursors, together with the relatively simple and versatile methods available for their synthesis.^41,42 In addition, the dissolution behaviour of metal oxide nanoparticles is strongly influenced by pH conditions.⁴³ Among various oxide nanoparticles, including titanium dioxide, chromium oxide, manganese oxide, iron oxide, nickel oxide, copper oxide (CuO), and zinc oxide, CuONPs and zinc oxide nanoparticles (ZnONPs) are of particular concern because the release of copper ion and zinc ion under physiological conditions may significantly contribute to their toxicity.⁴³

Although several review articles have explored the synthesis, biological applications, and toxicity of CuONPs, these studies are often focused on specific applications or presented in a fragmented manner. Much of the existing literatures focuses on the applications,^19,44–48 toxicity,^49–55 and even though some review focuses on both the application and the toxicity,^56,57 very few studies have mentioned the connection between physicochemical characteristics, exposure routes, biodistribution, pharmacological activity, and toxicity. Such an approach is important for better understanding both the promising applications and the safety considerations associated with CuONPs.

Copper Oxide Nanoparticles (CuONPs)

In this section, we will cover the synthesis, characterization and the physicochemical parameters of CuONPs.

CuONPs Synthesis

CuONPs can be synthesized using a variety of methods broadly classified into bottom-up and top-down approaches.⁵⁸ In the bottom-up approach, nanoparticles are built from atomic or molecular precursors using techniques such as the sol–gel method which utilizes a liquid precursor to produce the nanoparticles,⁵⁹ spinning method employs a spinning disc reactor for nanoparticle formation,⁶⁰ chemical vapor deposition facilitates nanoparticle synthesis through gas-phase reactions,⁶¹ and biologically mediated (green) synthesis which uses biological agents such as plant extracts or microorganisms.⁶² Conversely, top-down approach reduces bulk materials into nanoscale particles through physical processes such as mechanical grinding using ball milling,⁶³ sputtering using high energy ions to break down materials,⁶⁴ thermal decomposition involving the application of heat to induce fragmentation,⁶⁵ and laser ablation technique utilizing laser pulses for nanoparticles generation.⁶⁶ The synthesis of CuONPs is illustrated in Figure 2.

Figure 2.

Schematic representation of the synthesis approaches used for the preparation of copper oxide nanoparticles (CuONPs). (A) Bottom-up synthesis methods, where CuONPs are formed through nucleation and growth from atomic or molecular precursors, including the sol–gel method, chemical vapour deposition, spinning method, pyrolysis method, and green synthesis using plant extracts or microorganisms. (B) Top-down synthesis methods, where bulk copper oxide materials are reduced to nanoscale dimensions through physical processing techniques, including mechanical milling, sputtering, thermal decomposition, and laser ablation.

CuONPs Characterization and Physicochemical Properties

Characterization provides information on the physical and chemical properties of nanomaterials, enabling understanding of their structure, size, and morphology. A variety of analytical techniques are used to characterize CuONPs, including ultraviolet (UV) visible spectroscopy, X-ray diffractometry (XRD), scanning and transmission electron microscopy (SEM/TEM), Fourier transform infrared spectroscopy (FTIR), and zeta potential analysis. CuONPs typically exhibit a UV–visible absorption peak in the range of 200–440 nm.^67,68 XRD analysis confirms their crystalline nature, showing characteristic diffraction peaks corresponding to the monoclinic CuO structure.⁶⁹ SEM and TEM analyses reveal diverse morphologies, including flower-like, rod-shaped, spherical, and plate-like structures, with particle sizes generally ranging from ~19.6 to 104.6 nm depending on synthesis conditions.^70–72 FTIR spectra further confirm Cu–O bonding along with surface functional groups such as hydroxyl species.⁷³ Zeta potential values indicate colloidal stability and are strongly influenced by pH and the presence of surfactants. Higher absolute zeta potential values (positive or negative) are associated with improved dispersion stability by minimizing aggregation,⁷⁴ in the literatures it was reported that CuONPs may possess both negative⁷⁵ and positive charges.⁷⁶ Brunauer–Emmett–Teller (BET) analysis is used to determine surface area, which directly influences reactivity. Reported BET values for CuONPs vary widely depending on synthesis route, ranging from ~0.10–0.11 m²/g to as high as 52.6 m²/g for biogenically synthesized nanoparticles.^77,78 Thermal analysis such as thermogravimetric analysis/differential thermal analysis shows a multi-step decomposition process, including the removal of adsorbed water followed by decomposition of organic components. The final stage corresponds to calcination at approximately 500 °C for 2 h, resulting in the formation of stable CuONPs.⁷⁹

The surface functionalization modifies nanoparticles to enhance or add properties for medical use, using their inherent surface chemical groups as starting points.⁸⁰ Such modifications are particularly important because copper nanoparticles are inherently unstable under atmospheric conditions and are readily prone to oxidation.⁴² In addition, without appropriate stabilization strategies, nanoparticles tend to aggregate and undergo sedimentation, which can significantly reduce their functional efficacy and overall performance.^81,82 Furthermore, the loading efficiency of CuONPs is strongly influenced by nanoparticle–drug surface interactions. For example, a comparative study evaluating the adsorption of platinum-based anticancer drugs onto CuONPs demonstrated that cisplatin exhibited relatively high entrapment efficiency, whereas nedaplatin showed considerably lower loading efficiency.⁸³ This reduced entrapment efficiency was attributed to weaker interactions between nedaplatin and the CuONPs surface, highlighting that drug-specific surface interactions may limit the loading capacity and delivery performance of CuONPs.⁸³

It was identified that coating of CuONPs with nano silicates prevents aggregation and enhances the antibacterial activity.⁸⁴ The surface modification also has an effect over the colloidal stability. Polymer-based stabilizers such as sodium poly (4-styrene sulfonic acid-co-maleic acid) provide excellent stability in deionized water, maintaining dispersion with minimal aggregation for up to 30 days.⁸⁵ In contrast, stabilizers such as cetyltrimethylammonium bromide and polyvinylpyrrolidone (PVP) exhibit comparatively lower stability, leading to sedimentation over time.⁸⁵ However, under biologically relevant conditions, stability behaviour changes significantly. Studies evaluating reduced glutathione (GSH) and hyaluronic acid (HA) functionalized CuONPs in simulated biological fluids (saliva, plasma, gastric, and intestinal fluids) have shown that aggregation occurs in all media, accompanied by reduced zeta potential and, in some cases, copper ion release, particularly in gastric fluid.⁸⁶ Although HA-functionalized nanoparticles formed relatively smaller aggregates than GSH-modified ones, aggregation was not completely prevented.⁸⁶ Similarly, glutamic acid-functionalized CuONPs integrated into medical silicone tubes demonstrated broad-spectrum antibacterial activity, including effectiveness against multidrug-resistant bacteria.⁸⁷ Furthermore, coating with bovine serum albumin (BSA) reduces particle size, minimizes aggregation, and improves dispersion and stability.⁸⁸

Another physicochemical parameter that has an influence on the biological activity is the size of the nanoparticle. Owing to their nanoscale dimensions, relatively small nanoparticles can be internalized by cells and translocate across biological barriers; however, their uptake efficiency, intracellular trafficking and biodistribution depend on particle size, shape, surface properties and the biological system.^89,90 It is well established that the antibacterial activity of CuONPs is size-dependent, with smaller nanoparticles generally exhibiting greater efficacy against both Gram-positive and Gram-negative bacteria.^91,92 Smaller CuONPs (~20 nm) have been reported to show enhanced antibacterial activity compared to larger particles, likely due to their larger surface area and improved interactions with bacterial cell membranes.⁹² Whereas, in human bronchial epithelial cells BEAS-2B, the CuONPs demonstrated greater cellular uptake and a more uniform intracellular distribution than fine CuO particles, suggesting that particle size plays a critical role in nanoparticle internalization, intracellular trafficking, and the enhanced genotoxic activity associated with nanosized particles.⁹³ Particle shape also plays a role in biological interactions, with rod-shaped CuONPs showing higher bioaccumulation compared to spherical or platelet forms.⁹⁴ Additionally, surface charge significantly affects biological outcomes, as demonstrated in plant systems where negatively charged CuONPs reduced disease progression and enhanced biomass, whereas positively charged particles showed limited effect.⁹⁵ Furthermore, a conceptual framework illustrating the relationship between the physicochemical properties of CuONPs and their corresponding biological outcomes has been included in Figure 3.

Figure 3.

Influence of physicochemical parameters of copper oxide nanoparticles (CuONPs) on their biological outcomes. The biological activity and interactions of CuONPs are strongly governed by their physicochemical characteristics, including (1) surface functionalization, (2) particle size, (3) particle shape, and (4) surface charge. Surface functionalization prevents aggregation and enhances antibacterial performance through the incorporation of functional groups such as hydroxyl (–OH), amino (–NH₂), and carboxyl (–COOH) moieties. Particle size influences cellular uptake and antimicrobial efficacy, with smaller nanoparticles generally exhibiting enhanced biological activity. Particle shape affects cellular interactions, bioaccumulation, and tissue distribution, resulting in shape-dependent biological responses. Surface charge regulates electrostatic interactions with cells, proteins, and other biomolecules.

Routes of Exposure and Biodistribution

Nanoparticles can enter the body through several routes, including inhalation, dermal contact, and ingestion. Owing to their extremely small size, nanoparticles can reach and deposit in the alveolar regions of the lungs, where gas exchange occurs.^96,97 From there, they may cross the air–blood barrier, enter the bloodstream, and be transported to various organs such as the heart, liver, kidneys, pancreas, and spleen.^98,99 Besides inhalation, nanoparticles can also pass through the skin barrier¹⁰⁰ and even overcome the protective barriers of the eye.¹⁰¹ Ingestion represents another possible route through which nanoparticles may enter the body.¹⁰²

Upon exposure to biological environments, CuONPs readily form a protein corona through interaction with biomolecules. The composition and structure of this corona are governed by nanoparticle surface properties, including chemistry, porosity, and electrostatic characteristics.¹⁰³ This protein layer significantly influences nanoparticle functionality, potentially enhancing or reducing biological activity.¹⁰⁴ For example, CuONPs interacting with BSA form complexes stabilized via hydrogen bonding and van der Waals interactions without significantly altering protein structure.¹⁰⁵ In contrast, interaction with fibrinogen induces conformational changes, leading to protein misfolding, aggregation, and impaired coagulative function.¹⁰⁶ These findings indicate that nanoparticle–protein interactions are highly protein-specific.¹⁰⁶ Nanoparticles are primarily internalized via endocytosis.¹⁰⁷ During this process, nanoparticles interact with cell surface receptors, triggering membrane invagination and vesicle formation, ultimately leading to intracellular uptake.^108,109 Specifically, CuONPs have been shown to undergo dynamin-dependent endocytosis, followed by transport to endosomes and lysosomes, where they dissolve under acidic conditions and release copper ions, a mechanism commonly referred to as the “Trojan horse”.¹¹⁰ However, CuONPs may also undergo extracellular dissolution in biological fluids, resulting in the release of copper ions. These ions can subsequently be transported into cells through endogenous metal transport systems, including divalent metal transporter 1, thereby contributing to intracellular copper accumulation.^111,112 The different routes of exposure of CuONPs, along with their biodistribution and underlying mechanisms, are illustrated in Figure 4.

Figure 4.

Overview of the biodistribution and potential biological effects of copper oxide nanoparticles (CuONPs) following different exposure routes. CuONPs may enter the body via oral, inhalation, dermal, or intravenous routes. At the exposure site, CuONPs can trigger local responses through copper ion release, reactive oxygen species (ROS) production, and inflammation. After crossing physiological barriers such as the gastrointestinal mucosa, air–blood interface, and skin, nanoparticles may gain access to systemic circulation and subsequently accumulate in multiple organs, including the heart, brain, kidneys, liver, lungs, skin, testes, and developing embryo.

Respiratory Exposure

Among the various exposure routes, inhalation is considered the most significant pathway for unintentional human exposure to nanomaterials. Following inhalation, CuONPs deposit in the respiratory tract, particularly in the alveolar regions, where they may cross the air–blood barrier and enter systemic circulation, enabling their distribution to secondary organs.^36,113 Studies in both mice and rats have demonstrated that inhaled copper nanoparticles and CuONPs induce significant pulmonary toxicity, including lung inflammation, cytotoxicity, and tissue injury characterized by perivasculitis and alveolitis.^114,115 Histopathological analyses have further revealed degeneration of the nasal epithelium, bronchiolitis, emphysema, and vacuolation of the respiratory epithelium following exposure.¹¹⁵ Similarly, intratracheal (i.t.) instillation studies have reported dose-dependent fibrosis, granuloma formation, and lymphoid aggregation within lung tissues.¹¹⁶ Acute exposure to CuONPs has been shown to trigger inflammatory responses characterized by increased bronchoalveolar lavage fluid (BALF) white blood cell counts, elevated protein levels, and neutrophil infiltration in the lungs.¹¹⁷ This process is associated with the generation of reactive species through myeloperoxidase activation, alongside the release of inflammatory mediators such as interleukin (IL) - 1β and high mobility group box 1, and thereby contributing to tissue damage.¹¹⁷ Functionalized CuONPs have also been reported to induce dose-dependent pulmonary inflammation and cellular damage following inhalation.¹¹⁸ Accumulation of copper in lung tissues following inhalation of CuONPs has been observed over time, followed by gradual clearance, with an elimination half-life of approximately 6.5 days.¹¹³ In addition to local pulmonary effects, inhaled CuONPs exhibit the ability to translocate beyond the lungs.¹¹³ Notably, copper ions has also been detected in whole blood and heart tissue, indicating systemic distribution.¹¹³ Further evidence of systemic effects includes altered organ weights and disruption of trace element homeostasis in organs such as the kidneys and spleen.¹¹³ Similarly, a recent i.t instillation study demonstrated that pulmonary exposure to CuONPs extends beyond the lungs, inducing systemic effects including inflammation, oxidative stress, thrombosis, DNA damage and apoptosis.¹¹⁹ In addition to these systemic effects, nanoparticles including CuONPs have been reported to influence immune function by modulating dendritic cell maturation, antigen presentation, and downstream T-cell responses.^120,121 Chronic inhalation exposure results in time-dependent systemic immune alterations, characterized by early activation of T-cell responses followed by transient suppression, along with fluctuations in lymphocyte populations and modulation of cytokine production over time.¹²²

Oral Exposure

CuONPs are increasingly used in medical applications and food-packaging systems due to their antifouling, antimicrobial, antioxidant, and biosensing properties.^123,124 Despite these advantages, their incorporation into packaging materials has raised important safety concerns. Several studies have demonstrated that nanoparticles can migrate from packaging into food, thereby creating a potential route for oral exposure.¹²⁵ Metal nanoparticles used in food packaging can migrate into food upon direct contact, often through dissolution into ionic species.¹²⁶ For instance, copper migration into chicken breast has been reported at levels ranging from 0.024–0.049 mg/dm², with storage time and temperature showing no significant effect on migration.¹²⁷ Similarly, studies investigating the oral exposure of copper nanoparticles have demonstrated that they undergo high dissolution under gastric conditions, facilitating the release of copper ions that subsequently distribute through the bloodstream to various organs, particularly the liver, kidneys, and spleen, thereby contributing to organ-specific toxicity.¹²⁸ Furthermore, copper nanoparticles were not completely absorbed following oral administration, with a substantial proportion being eliminated through faecal excretion.¹²⁸ In addition, oral exposure to CuONPs has been shown to induce intestinal inflammation, impair intestinal barrier integrity, and disrupt bile acid homeostasis, thereby contributing to the development of non-alcoholic fatty liver disease.¹²⁹ These adverse gastrointestinal effects are further supported by in vitro studies using both two-dimensional rat intestinal and three-dimensional human intestinal cell models which demonstrated that CuONPs exert cytotoxic effects, with ROS generation playing a key role in inducing oxidative stress and cellular damage.¹³⁰

Skin Exposure

Human exposure to CuONPs may also occur through dermal contact. The epidermis functions as an effective barrier, with intact skin showing negligible permeation of copper ions. However, disruption of the stratum corneum can facilitate the penetration of nanoparticles into deeper skin layers.¹³¹ Most studies indicate that nanoparticles show limited skin penetration, often remaining within or on the stratum corneum, evidence regarding deeper penetration remains unclear.^132,133 Some studies have reported that certain nanoparticles can penetrate intact and damaged human skin and reach deeper layers such as the dermis; however, this penetration is highly dependent on nanoparticle properties and skin condition.^134,135 In a study using a Franz static diffusion model on human skin, CuONPs exhibited negligible absorption through intact skin; however, significantly increased permeation of copper was observed when the skin barrier was compromised.¹⁰⁰ Furthermore, the ability of nanoparticles to penetrate the skin is strongly size-dependent.¹³⁶ Particles ≤ 4 nm can penetrate intact skin, those between 4–20 nm may penetrate both intact and damaged skin, while larger particles (21–45 nm) generally penetrate only damaged skin, and particles >45 nm are unlikely to penetrate the skin barrier.¹³⁶ For metal-based nanoparticles, including CuONPs, penetration may be associated with partial dissolution into metal ions within the skin, contributing to local and potentially systemic effects.¹³⁶ At the cellular level, CuONPs have been shown to internalize within human epidermal cells, leading to cytotoxic and genotoxic effects.¹³⁷ These effects are largely mediated by the generation of ROS, resulting in oxidative stress and triggering cell death through both apoptosis and necrosis.¹³⁷

Parenteral Exposure

Parenteral administration provides a direct route for CuONPs to bypass physiological barriers and enter systemic circulation. Once in the bloodstream, these nanoparticles can distribute to various tissues and cells based on their ability to diffuse across them.⁵² Parenteral administration of CuONPs based drug formulations has demonstrated enhanced antipyretic, analgesic, and anti-inflammatory effects, primarily due to the improved delivery and efficacy of loaded therapeutic agents.¹³⁸ On the other hand subchronic intraperitoneal (i.p.) administration of CuONPs has been shown to induce organ-specific toxicity, particularly affecting the liver, spleen, kidneys, and brain.¹³⁹ These toxic effects are associated with alterations in biochemical parameters, including increased lactate dehydrogenase (LDH) activity, elevated lipid peroxidation, and depletion of intracellular GSH, reflecting oxidative stress. Moreover, changes in systemic markers such as creatinine and transaminases eg, serum glutamic oxaloacetic transaminase (SGOT) are indicative of organ-specific toxicity.^140–142 It was observed that following i.p. administration in mice, both chemically synthesized and green-synthesized CuONPs accumulated primarily in the liver and spleen, with subsequent distribution to the kidneys, lungs, heart, and intestine.¹⁴⁰ However, chemically synthesized CuONPs showed greater accumulation in the liver and other organs, whereas green-synthesized CuONPs exhibited higher systemic availability and more efficient faecal excretion, suggesting improved clearance from the body.¹⁴⁰ Similarly, intravenous (i.v.) administration of elesclomol-loaded CuONPs resulted in rapid tumour accumulation in melanoma-bearing mice, with detectable localization at the tumour site within 1 h post-injection.¹⁴³ The nanoparticles also exhibited prolonged tumour retention, persisting for up to 24 h. In addition to tumour localization, elesclomol-loaded CuONPs were distributed to major organs, including the liver, spleen, kidney, lungs, and heart, indicating systemic biodistribution following i.v. exposure.¹⁴³

Pharmacological Activities of CuONPs

Antibacterial Activity of CuONPs

One of the most important activities attributed to CuONPs is their antibacterial potency. In the following sections, the discussion is based on the antibacterial activity of both chemically and green synthesized CuONPs.

Antibacterial Activity of Chemically Synthesized CuONPs

The antibacterial efficacy of chemically synthesized CuONPs is highly dependent on many factors like the oxidation state, availability of releasable copper ions, doping nature along with interaction of CuONPs with other materials which will be discussed in the following paragraph.

For instance, it was observed that CuONPs with an oxidation state of +2 exhibited comparatively higher antibacterial activity than those with an oxidation state of +1. In this context, Asamoah et al¹⁴⁴ observed that the +2 oxidation state of the CuONPs showed better antibacterial efficacy compared to other metal oxide nanoparticles like ZnONPs in both gram positive and gram negative bacteria’s. In contrast, Uthra et al¹⁴⁵ reported that CuONPs with an oxidation state of +1 showed lesser efficacy as an antibacterial agent when compared with ZnONPs against biofilm producing multi-drug-resistant organisms. However, this effect is heavily attributed towards the varying bacterial strains and bacterial biology.^144,145 Furthermore, when a mixed oxidative system of the CuONPs were prepared, then together as a system, they could tackle many food borne pathogens with relatively higher antibacterial efficacy.¹⁴⁶ However, the study did not compare the effect of the +1 and +2 oxidation states therefore, additional work is needed to clarify this point.¹⁴⁶ Gram-positive and Gram-negative bacteria resistant to β-lactam antibiotics such as methicillin and carbapenems were effectively inhibited by CuONPs supported zeolite.¹⁴⁷ Nevertheless, although the CuONPs supported material exhibited antibacterial activity, zeolite saturated with copper ions alone demonstrated superior bactericidal performance, as indicated by lower minimum bactericidal concentration values, despite similar minimum inhibitory concentration (MIC) values.¹⁴⁷ The superior antibacterial activity of ion-saturated zeolites can be attributed to the greater availability of free copper ions, which readily interact with bacterial cells.¹⁴⁷ In contrast, CuONPs supported zeolites exhibited lower antibacterial efficacy, suggesting that direct copper ion availability plays a key role in antibacterial action.¹⁴⁷ These findings indicate that the antibacterial effect is largely governed by the release and activity of copper ions.¹⁴⁷ Another study reported by Khlifi et al¹⁴⁸ demonstrated that doping CuONPs with transition metals enhances antibiofilm activity. Among the dopants, manganese (Mn) doped CuONPs exhibited high efficiency at lower concentrations, whereas iron and nickel doped CuONPs showed improved activity at higher concentrations.¹⁴⁸ Overall, the doped CuONPs demonstrated superior performance compared to undoped CuONPs.¹⁴⁸ This variation highlights the critical role of the dopant type in determining antibiofilm activity, which may be attributed to differences in oxygen absorption as enhanced oxygen interactions can promote increased antibacterial activity.¹⁴⁸ The interfacial interaction of CuONPs with other materials like graphene oxide (GO) can also play a significant role in the antibacterial activity, as this interaction of CuONPs with GO showed a broad-spectrum activity towards both gram positive and gram-negative bacteria with prominent effect on the gram-negative bacteria.¹⁴⁹ From this we may also understand that the antibacterial activity is influenced by factors like nature of the interacting materials with the bacterial cell membranes.¹⁴⁹

Antibacterial Activity of Green Synthesized CuONPs

Green synthesis has emerged as an effective and environmentally friendly approach to produce CuONPs with better antibacterial properties. The use of plant extracts not only facilitate the nanoparticle formation but also introduces bioactive compounds that can have an influence on their stability and biological activity. The antibacterial activity of green synthesized CuONPs depends on many factors like the capping materials, the mode of action, dose of the nanoparticles and the biomolecular composition which will be discussed in the following paragraphs.

Khairy et al¹⁵⁰ reported that CuONPs synthesized using ethanolic neem extract exhibited a larger zone of inhibition against Klebsiella species compared to those synthesized using jojoba extract. However, in terms of antibiofilm activity, the jojoba-mediated CuONPs demonstrated superior inhibition compared to neem-derived nanoparticles.¹⁵⁰ This variation can be attributed to differences in the capping phytochemicals present on the nanoparticle surface, which influence their properties.¹⁵⁰ Similarly, in another study,⁶⁸ it was identified that CuONPs synthesized from the dried fruit of Terminalia chebula exhibited the highest zone of inhibition against Pseudomonas aeruginosa. However, in terms of antibiofilm activity, Staphylococcus aureus showed the highest susceptibility, while Pseudomonas aeruginosa exhibited comparatively greater resistance.⁶⁸ From this, it can be concluded that the antibacterial efficacy of CuONPs depends not only on the type of bacterial species but also on the mode of activity being evaluated, such as planktonic like growth inhibition versus biofilm disruption.⁶⁸ The antibacterial efficacy of CuONPs synthesized from Silybum marianum was found to increase with concentration, as evidenced by the marked enhancement in the zone of inhibition when the concentration was increased from 4 mg/mL to 20 mg/mL.¹⁵¹ This indicates a clear dose-dependent antibacterial effect, suggesting that nanoparticle concentration plays a crucial role in determining their antimicrobial activity.¹⁵¹ The nanoparticle efficacy also depends on the biological source used for green synthesis. While previous studies primarily focused on plant-mediated CuONPs, fungal-mediated synthesis using Ganoderma sessile demonstrated distinct behavior.⁷⁵ In this study, nanoparticle synthesized from the supernatant exhibited lower MIC values, indicating enhanced antibacterial activity, whereas extract-mediated nanoparticles showed differences in half maximal inhibitory concentration (IC₅₀) values, particularly against Pseudomonas aeruginosa.⁷⁵ This variation can be attributed to differences in the biomolecular composition of the supernatant and extract, which act as capping agents and influence nanoparticle surface chemistry.⁷⁵

Mechanism of Antibacterial Activity

Copper containing nanoparticles can readily dissolve under physiological conditions, resulting in the release of metal ions that can directly interact with bacterial cell membranes.^152,153 Furthermore, the positively charged surface of metal oxide nanoparticles promotes electrostatic interactions with negatively charged bacterial membranes, facilitating nanoparticle adhesion and cellular internalization. These interactions can disrupt membrane integrity, induce leakage of intracellular components, and ultimately lead to bacterial cell death.^146,154,155 In addition, CuONPs have been shown to inhibit biofilm formation, reduce swarming motility, and decrease adenosine triphosphate (ATP) production in bacterial cells.¹⁵⁶ Following internalization, CuONPs stimulate the generation of ROS, particularly superoxide radicals, which cause oxidative damage to cellular components including membranes, proteins, lipids, and DNA, thereby contributing to bacterial cell death.^157,158 A schematic illustration of the antibacterial mechanism of CuONPs is presented in Figure 5.

Figure 5.

Proposed antibacterial mechanism of copper oxide nanoparticles (CuONPs). CuONPs exert antibacterial activity through both ion-mediated and particle-mediated mechanisms. Upon dissolution, CuONPs release copper (Cu²⁺) ions, which penetrate bacterial cells and promote the generation of reactive oxygen species (ROS). Simultaneously, intact CuONPs can be internalized by bacterial cells, where they undergo dissolution in acidic intracellular compartments, releasing additional Cu²⁺ ions. These ions participate in Fenton-like reactions, producing highly reactive radicals that further enhance oxidative stress. The excessive accumulation of ROS results in deoxyribonucleic acid (DNA) damage, including strand breaks, base oxidation, and mutations; protein oxidation leading to loss of function; and mitochondrial damage, including disruption of membrane potential and electron transport.

Anticancer Activity of CuONPs

This section deals with the in vitro anticancer activity of both chemically and green synthesized CuONPs.

Anticancer Activity of Chemically Synthesized CuONPs

The anticancer potential of CuONPs is attributed to their dose-dependent cytotoxic effects, variability in selectivity toward cancer and normal cells, and their dependence on nanoparticle characteristics and experimental conditions which will be discussed in the following paragraph.

The CuO/GO nanocomposite exhibited a clear dose-dependent cytotoxic effect against epidermoid carcinoma cell line A-431, with increasing concentrations leading to a concomitant reduction in cell viability.¹⁴⁹ Although the study evaluated cytotoxicity at two time points (24 h and 48 h), only a marginal additional decrease in viability was observed at 48 h.¹⁴⁹ This suggests that the cytotoxic response is predominantly concentration-driven, with minimal influence of exposure time under the tested conditions.¹⁴⁹ Chemically synthesized CuONPs have been shown to induce significant cytotoxic effects in hepatocarcinoma cell line HepG2.¹⁵⁹ In contrast, CuONPs could show potential toxicity and apoptosis in human chronic myelogenous leukaemia cell line K-562 primarily through ROS generation serving as a potential anti-cancer agent as it did not kill the healthy normal cells.¹⁶⁰ These contrasting findings suggest that the cytotoxic behaviour of chemically synthesized CuONPs is highly dependent on experimental conditions, cell type, and nanoparticle characteristics.¹⁶⁰ In a study of Mn doped CuONPs, the cytotoxic response was both dose- and composition-dependent.¹⁶¹ CuO: Mn3 induced a more variable and less selective cellular response, whereas CuO: Mn4 exhibited greater biocompatibility toward normal fibroblasts while maintaining pronounced cytotoxic effects against melanoma cells at higher concentrations.¹⁶¹ These findings suggest that Mn doping strongly influences the biological activity and selectivity of CuONPs.¹⁶¹ However, this effect is not simply dependent on increasing dopant concentration. Instead, both the level of Mn doping and the nanoparticle dose together determine whether the nanoparticles are toxic to cells or remain biocompatible.¹⁶¹

Anticancer Activity of Green Synthesized CuONPs

The anticancer activity of CuONPs depends on various parameters such as calcination temperature, synthesis approach, nanoparticle composition, and concentration, which influence their cytotoxicity and selectivity toward cancer cells.

In a study investigating calcined CuONPs prepared at different temperatures (400, 500, 700, and 900 °C), all samples exhibited time- and dose-dependent cytotoxicity.¹⁶² Notably, CuONPs calcined at 900 °C demonstrated reduced toxicity toward normal human embryonic kidney cell line HEK-293, while maintaining significant cytotoxic effects against human colorectal cell line HCT-116 and human breast cancer cell line MCF-7, indicating improved selectivity at higher calcination temperatures.¹⁶² Similarly, CuONPs synthesized using Camellia sinensis extract were evaluated against human colon cell line HT-29 and MCF-7, where they were shown to induce cell cycle arrest and apoptosis.¹⁶³ Although the nanoparticles were described as biocompatible and exhibited moderate selectivity toward normal cells, the difference in IC₅₀ values between cancerous and normal cells was relatively small.¹⁶³ This limited disparity suggests that the selectivity of the nanoparticles may be insufficient, raising concerns about their potential toxicity to healthy cells.¹⁶³ In contrast, a copper/copper(II) oxide/copper(I) oxide nanoparticle composite demonstrated dose-dependent cytotoxicity against MCF-7 cells, with cell viability decreasing as concentration increased.¹⁶⁴ However, this effect reflects general cytotoxicity rather than cancer-specific activity, as the study did not include comparisons with normal cell lines to evaluate selectivity.¹⁶⁴ Furthermore, a separate study reported that CuONPs synthesized using pumpkin seed extract induced a dose-dependent decrease in cell viability in the human breast cancer cell line MDA-MB-231.¹⁶⁵ This effect was accompanied by notable morphological changes, including cell shrinkage, detachment, membrane blebbing, and cellular deformation.¹⁶⁵ Phyto-fabricated CuONPs have also been shown to exert cytotoxic effects in HepG2 cells.¹²⁴ However, studies have reported that this effect is not entirely selective, as normal cells are also affected.¹²⁴ In line with this, dose-dependent analyses demonstrate that higher nanoparticle concentrations enhance cancer cell inhibition but also increase toxicity toward normal cells.¹²⁴ Although surface modifications such as chitosan coating can improve anticancer efficacy, they do not eliminate the impact on healthy cells.¹²⁴ However, the exact mechanism responsible for the non-selective cytotoxicity was not explicitly explored in the study.¹²⁴

Mechanism of Anticancer Activity of CuONPs

The anticancer activity of CuONPs is primarily mediated through the induction of oxidative stress. Following cellular internalization, CuONPs stimulate the excessive generation of ROS and nitric oxide (NO).¹⁶⁶ Increased NO levels can react with ROS to form highly reactive nitrogen species, such as peroxynitrite, resulting in oxidative damage to cellular macromolecules, including DNA, proteins, lipids, and mitochondria.¹⁶⁶ This cellular stress activates tumour suppressor pathways, as evidenced by the upregulation of p53 and its downstream pro-apoptotic target Bcl – 2- associated X protein (BAX).¹⁶⁷ The increased expression of p53 and BAX promotes activation of the intrinsic mitochondrial apoptotic pathway, ultimately leading to programmed cell death.¹⁶⁷ Concurrently, CuONPs downregulate the expression of the anti-apoptotic gene B-cell lymphoma 2, thereby suppressing cellular survival mechanisms and further enhancing apoptosis.¹⁶⁶ In addition, the downregulation of cyclin D1, a key regulator of cell-cycle progression, inhibits cellular proliferation by inducing cell-cycle arrest.¹⁶⁷ Moreover, CuONPs can also trigger early oxidative stress response.¹⁶⁸ The schematic illustration highlighting the mechanism of anticancer activity of CuONPs is presented in Figure 6.

Figure 6.

Schematic mechanism of copper oxide nanoparticles (CuONPs) induced cytotoxicity and apoptosis in cancer cells. CuONPs exert their cytotoxic effects through both extracellular dissolution and cellular internalization pathways. Following dissolution, CuONPs release copper (Cu²⁺) ions, which contribute to the generation of reactive oxygen species (ROS). Alternatively, CuONPs can be internalized via endocytosis and trafficked to lysosomes, where acidic conditions promote further Cu²⁺ ion release. Moreover, it was also identified that oxidative damage can occur even before detectable lysosomal dissolution. Elevated ROS levels induce oxidative stress, leading to DNA damage resulting in increased expression of p53 and pro-apoptotic protein Bcl-2-associated X protein (BAX), decreased cyclin D1 (CCND1) expression, and subsequent cell-cycle arrest. Moreover, the BAX protein translocated and forms pores in the mitochondria leading to mitochondrial apoptosis.

Antidiabetic Activity of CuONPs

This section deals with the antidiabetic activity of both chemically and green synthesized CuONPs.

Antidiabetic Activity of Chemically Synthesized CuONPs

The antidiabetic potential of chemically synthesized CuONPs appears to be influenced by their capping agents and surface modifications.

When CuONPs were synthesized using a Schiff base (SB) as a capping agent, their antidiabetic potential was evaluated through α-amylase and α-glucosidase inhibitory assays.¹⁶⁹ The SB-CuONPs exhibited 51.03% inhibition of α-amylase and 42.82% inhibition of α-glucosidase, indicating their ability to inhibit carbohydrate-digesting enzymes and suggesting potential antidiabetic activity.¹⁶⁹ Similarly, when capping agents such as PVP and polyethylene glycol (PEG) were incorporated into CuONPs, both capped CuONPs demonstrated significant α-amylase inhibitory activity.¹⁷⁰ Among them, CuO-PVP nanoparticles exhibited greater inhibition (37.75%) compared to CuO-PEG nanoparticles, which showed 32.23% inhibition.¹⁷⁰ These findings suggest that the nature of the capping agent plays an important role in modulating the inhibition of carbohydrate-digesting enzymes.

Antidiabetic Activity of Green Synthesized CuONPs

The antidiabetic potential of green synthesized CuONPs can be attributed to factors like synthesis method, plant phytochemical composition along with the physicochemical parameters like calcination and surface modification which will be discussed in the following paragraphs.

Umar et al¹⁷¹ demonstrated that CuONPs synthesized from Cereus hildmannianus significantly reduced blood glucose levels in alloxan-induced diabetic Wistar rats at a dose of 300 mg/kg body weight. After 21 days of treatment, the blood glucose concentration declined to below 200 mg/dL, an effect comparable to that produced by the standard antidiabetic drug glibenclamide.¹⁷¹ Similarly, CuONPs prepared using Bacopa monnieri leaf extract exhibited strong antidiabetic effects in streptozotocin-induced diabetic mice, producing a 35.74% reduction in blood glucose when administered alone and a 32.78% reduction in the group receiving both CuONPs and insulin.¹⁷² These findings collectively suggest that biosynthesized CuONPs may exert glucose-lowering effects through mechanisms comparable to conventional antidiabetic agents.¹⁷² In another study CuONPs synthesized from Opuntia ficus leaf extract strongly inhibited α-amylase and α-glucosidase, showing inhibition values of 91.5% and 82.3%, respectively, at the highest concentration (1 mg/mL).¹⁷³ At the lowest concentration tested (0.0019 mg/mL), inhibition decreased to 34.1% and 32.35%, indicating a shift from strong to mild enzyme inhibition as dose decreased.¹⁷³ Notable enzyme inhibition was also observed using CuONPs synthesized from the Psidium guajava varieties Allahabad Safeda and Hisar Safeda.¹⁷⁴ In the α-amylase assay, CuONPs from the Allahabad Safeda variety produced 67.32% inhibition, whereas the Hisar Safeda variety yielded 63.25%, indicating stronger inhibitory activity for the former.¹⁷⁴ A similar trend was observed in the α-glucosidase assay, with inhibition values of 75.18% for the Allahabad Safeda CuONPs and 71.38% for the Hisar Safeda CuONPs.¹⁷⁴ This variation suggests that differences in plant phytochemical composition may influence and alter nanoparticle biological activity.¹⁷⁴ Beyond enzyme inhibition, Zidane et al¹⁷⁵ investigated glucose uptake in yeast cells using calcined and non-calcined CuONPs synthesized from Algerian propolis. Their results showed that non-calcined CuONPs produced a pronounced decrease in extracellular glucose levels as nanoparticle concentration increased, reflecting strong glucose uptake.¹⁷⁵ These findings imply that physicochemical factors such as calcination and surface modification may significantly influence the antidiabetic efficacy of CuONPs.¹⁷⁵

Mechanism of Antidiabetic Activity of CuONPs

The antidiabetic action of CuONPs is largely attributed to their capacity to inhibit α-amylase and α-glucosidase, two key enzymes involved in carbohydrate digestion and subsequent glucose absorption.^151,173 Under normal physiological conditions, dietary polysaccharides are initially broken down by pancreatic α-amylase into smaller oligosaccharides, which are then further hydrolysed into glucose by α-glucosidase at the intestinal brush border.¹⁷⁶ The released glucose is subsequently transported into the bloodstream via glucose transporters and sodium–glucose co-transporters, contributing to postprandial hyperglycaemia.^177–180 By inhibiting these digestive enzymes, CuONPs slow the conversion of complex carbohydrates into glucose and limit its intestinal uptake.^173,174

Antioxidant Activity of CuONPs

This section discusses the antioxidant activity of both chemically and green synthesized CuONPs.

Antioxidant Activity of Chemically Synthesized CuONPs

The antioxidant activity of chemically synthesized CuONPs is strongly influenced by factors such as synthesis method, nanocomposite formation, and surface modification, all of which can alter their radical scavenging efficiency and overall antioxidant potential.

CuO/GO nanocomposites prepared from copper nitrate and citric acid, with GO added intermittently during synthesis, exhibited notable antioxidant activity in both 2,2′-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) and 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging assays, with IC₅₀ values of 55.70 ± 0.19 μg/mL and 44.86 ± 1.74 μg/mL, respectively.¹⁴⁹ The enhanced antioxidant activity observed in these nanocomposites may be attributed to the incorporation of GO.¹⁴⁹ Similarly, CuONPs synthesized via the sol–gel method demonstrated concentration-dependent antioxidant activity in DPPH and hydroxyl radical scavenging assays, with scavenging efficiencies increasing from 9% to 62% and 9% to 72%, respectively, across concentrations ranging from 15 to 500 μg/mL.¹⁸¹ However, the lower scavenging efficiency compared with ascorbic acid under identical conditions suggests that, although CuONPs possess antioxidant potential, their activity remains less potent than conventional antioxidant standards.¹⁸¹ CuONPs produced through thermal decomposition also displayed strong antioxidant behaviour in the DPPH assay, achieving a 85% scavenging rate within 1 h.¹⁸² The rapid reduction in DPPH absorbance indicates efficient free radical neutralization, suggesting that synthesis methods involving thermal decomposition may improve antioxidant performance.¹⁸² Surface modification further appears to play a significant role in modulating antioxidant activity.¹⁷⁰ CuONPs synthesized through a co-precipitation route and coated with PEG or PVP, PVP-coated nanoparticles exhibited the highest phenolic and flavonoid contents, together with superior total antioxidant activity and reducing power with DPPH radical scavenging activity of 28.36%, whereas PEG-coated CuONPs demonstrated greater DPPH radical scavenging activity of about 34.14%, whereas the uncapped CuONPs showed an activity of only 13.79%.¹⁷⁰ These findings indicate that different capping agents may influence antioxidant mechanisms differently and the activity with capping agents are more compared to the uncapped CuONPs.¹⁷⁰

Antioxidant Activity of Green Synthesized CuONPs

The antioxidant activity of biosynthesized CuONPs has attracted considerable attention due to their ability to scavenge ROS and modulate cellular antioxidant defence mechanisms which will be discussed in the paragraph.

Biosynthesized CuONPs have repeatedly demonstrated stronger antioxidant activity than those produced through conventional chemical routes. In the DPPH assay, CuONPs derived from Solanum nigrum showed inhibition ranging from 19% to 90% at concentrations between 15 and 500 μg/mL, with an IC₅₀ of 131.54 μg/mL.¹⁸¹ Their hydroxyl radical scavenging activity followed a similar trend, producing 17–93% inhibition across the same concentration range.¹⁸¹ The concentration-dependent increase in scavenging activity suggests enhanced free radical neutralization at higher nanoparticle concentrations.¹⁸¹ CuONPs prepared from Cissus vitiginea leaves were also evaluated for DPPH scavenging, in which the characteristic purple colour of DPPH gradually faded with increasing nanoparticle concentration.¹⁸³ These nanoparticles recorded an IC₅₀ of 45.29 μg/mL, a value close to that of ascorbic acid (IC₅₀ = 41.33 μg/mL), indicating comparatively strong antioxidant potential among biosynthesized CuONPs.¹⁸³ To further assess their influence on enzymatic antioxidant systems, green-synthesized CuONPs have been examined using superoxide dismutase (SOD) and catalase (CAT) assays the two enzymes central to protecting plant cells from oxidative stress.¹⁸⁴ In studies involving tomato and cauliflower plants, SOD activity increased by 60–61% at a moderate dose of 100 mg/L, reflecting an early protective response.¹⁸⁵ At a higher concentration of 500 mg/L, SOD activity declined, likely due to enzyme inhibition or excessive stress.¹⁸⁵ In contrast, CAT activity reached its maximum at this higher concentration, rising by 221% in cauliflower and 189% in tomato, suggesting that CAT becomes the dominant antioxidant enzyme when SOD activity begins to diminish.¹⁸⁵

Pathophysiological Effects of CuONPs

The pathophysiological effects of CuONPs describe how these particles interact with biological systems, causing changes at both the cellular and whole-organism levels that may disrupt normal function and potentially contribute to disease.

In vitro Effects

The in vitro biological effects of CuONPs have been extensively studied, and the key findings are summarized in the following sections. Nanoparticles can mediate toxicity through oxidative stress, inflammation, genetic damage and cell death.^186,187

CuONPs Induced Oxidative Stress

ROS are natural by-products of aerobic cellular metabolism and are primarily generated during mitochondrial respiration.¹⁸⁸ Oxidative stress occurs when the production of ROS exceeds the body’s antioxidant defence capacity, resulting in an imbalance between oxidant generation and detoxification mechanisms.¹⁸⁹ ROS can be beneficial to the organisms as cell regulators, but in higher doses, they can be cytotoxic which can eventually lead to cell death.^190,191

Oxidative stress was assessed in adult Fasciola hepatica worms exposed to CuONPs at concentrations of 1, 4, 8, 12, and 16 ppm.¹⁹² Exposure to 8–16 ppm CuONPs resulted in a concentration-dependent increase in ROS production, accompanied by reductions in SOD, GSH, and Glutathione S-transferase (GST) activities and an increase in malondialdehyde (MDA) levels, indicating oxidative stress.¹⁹² However, the precise molecular mechanisms underlying CuONPs induced oxidative stress were not investigated.¹⁹² CuONPs induced significant oxidative stress in human airway epithelial cell lines HEp-2 in a concentration-dependent manner.¹⁹³ Exposure to CuONPs at 10, 20, and 40 μg/mL for 24 h resulted in a progressive increase in lipid peroxidation levels, reaching 197% at the highest concentration.¹⁹³ Conversely, intracellular GSH levels decreased in a dose-dependent manner, with a maximum reduction of 66% observed at 40 μg/mL.¹⁹³ These findings indicate that CuONPs disrupt the cellular redox balance by enhancing oxidative damage and depleting antioxidant defences, thereby contributing to cellular toxicity.¹⁹³ In a similar cell line of HEp-2, CuONPs exposure significantly increased intracellular ROS generation, as evidenced by a 131% increase in 2′,7′-dichlorofluorescein fluorescence.¹⁹⁴ This oxidative burden was accompanied by impairment of the cellular antioxidant defence system, including a 25% reduction in CAT activity and a 29% decrease in glutathione reductase activity.¹⁹⁴ Although both studies employed the same HEp-2 cell model and demonstrated CuONPs induced oxidative stress, they differed in the oxidative stress biomarkers assessed.^193,194 In another study investigating the effects of CuONPs on human umbilical vein endothelial cell line HUVEC, exposure to CuONPs significantly increased intracellular superoxide anion levels, indicating excessive ROS generation.³⁶ In response to this oxidative challenge, antioxidant defence mechanisms were activated, as evidenced by the upregulation of antioxidant proteins such as heme oxyenase - 1 and glutamate cysteine ligase modifier subunit.³⁶ However, the cellular antioxidant capacity was progressively depleted, reflected by a reduced GSH/oxidized glutathione ratio.³⁶ Furthermore, CuONPs exposure promoted the accumulation of MDA, a marker of lipid peroxidation, indicating oxidative damage to cellular membranes.³⁶ Collectively, these findings demonstrate that CuONPs disrupt cellular redox homeostasis, leading to oxidative stress-mediated endothelial injury and subsequent cellular dysfunction.³⁶ In a study using HEK-293, exposure to CuONPs at concentrations of 10–300 µg/mL resulted in a concentration-dependent increase in LDH release, indicating cell membrane damage and cytotoxicity.¹⁹⁵ This was accompanied by a significant reduction in intracellular GSH levels, suggesting depletion of antioxidant defences.¹⁹⁵ However, ROS production was not directly measured in this study; therefore, the involvement of oxidative stress was inferred from secondary markers such as GSH depletion and LDH leakage rather than direct ROS quantification.¹⁹⁵

Inflammatory Responses

Inflammatory responses are protective biological reactions of the immune system activated by stimuli such as cellular damage, toxic substances, or pathogenic agents.¹⁹⁶ Acute inflammation generally plays a vital role in host defense, healing and tissue repair; however, an excessive or dysregulated acute inflammatory response can itself cause tissue damage and contribute to disease. Chronic inflammation is associated with several diseases, including autoimmune disorders, cardiovascular dysfunction and cancer.^197–199

Exposure of murine alveolar macrophages cell line MH-S to CuONPs activated inflammatory responses, as evidenced by the upregulation of cytokine-related pathways and increased expression of IL-17A.³⁰ Mechanistically, this effect was mediated through ferredoxin 1 (FDX1) dependent cuproptosis, linking copper-induced mitochondrial dysfunction to the initiation of inflammatory signalling.³⁰ Heterophils are immune cells found in birds like chickens. When these cells are isolated and cultured and when increasing concentrations of CuONPs were added to the cells, it was found that CuONPs could trigger the release of heterophil extracellular trap formation which are weblike structures or extracellular traps released by heterophils in a dose-dependent manner.²⁰⁰ Extracellular traps are triggered by inflammatory stimuli and have been shown to correlate positively with established inflammatory markers such as C- reactive protein and erythrocyte sedimentation rate, suggesting a close association between trap formation and inflammatory responses.^201,202 The inflammatory effects of CuONPs are influenced by both their synthesis method and the site of exposure.²⁰³ In this context, it can be reported that sonochemically synthesized CuONPs which are produced using ultrasonic wave-assisted chemical reactions, and commercially available CuONPs in human colorectal adenocarcinoma cell line Caco-2, differential cytokine responses were observed between apical and basolateral compartments.²⁰³ Sonochemically synthesized CuONPs induced IL-8 release only at a higher concentration (100 µg/mL) following apical exposure, whereas both nanoparticle types increased IL-8 levels upon basolateral exposure.²⁰³ In contrast, IL-6 exhibited only a marginal increase at 100 µg/mL under basolateral conditions with commercial CuONPs, while tumour necrosis factor-alpha levels remained unchanged across all conditions.²⁰³ These findings suggest that CuONPs induced inflammatory responses are both compartment- and cytokine-specific, highlighting the importance of exposure route and nanoparticle characteristics in modulating cellular responses.²⁰³ In a different study CuONPs induced IL-8 production in both differentiated and undifferentiated Caco-2 cells; however, the response was significantly greater in undifferentiated cells, indicating increased sensitivity compared to the more physiologically relevant differentiated model.²⁰⁴

Genotoxicity

Genotoxicity is mentioned as the ability of chemicals to cause damage in the genetic information of the body which can result to mutations and malignancies.²⁰⁵ The latter can be a consequence of both oxidative stress and inflammatory responses. The following section will cover the genotoxic potential of the CuONPs.

CuONPs when exposed to fibroblast like cell line RTG-2 which is derived from the reproductive tissue of rainbow trout, showed an increase in the tail DNA which is a measure of DNA damage in cells obtained using comet assay.²⁰⁶ Green-synthesized CuONPs were evaluated for genotoxicity using a chromosomal aberration assay in Allium cepa root cells.²⁰⁷ At higher concentrations (750 and 1000 μg/mL), CuONPs exhibited significant genotoxic effects, including acentric chromatin formation, chromatid breakage, and chromosomal fragmentation.²⁰⁷ In contrast, lower concentrations (250 and 500 μg/mL) did not induce observable chromosomal abnormalities.²⁰⁷ These findings indicate a clear dose-dependent genotoxic effect of CuONPs.²⁰⁷ In another study, the genotoxic potential of CuONPs against neuroblastoma cell line Neuro-2A was studied and it was identified that when CuONPs was exposed at a concentration of 50 mg/L, there was increased DNA fragmentation whereas when the concentration of CuONPs was 100 mg/L, then, there was a reduced DNA fragmentation.²⁰⁸ Although both studies demonstrated the genotoxic potential of CuONPs, the differences in response patterns may be attributed to variations in biological models, the dosage used and the genotoxicity endpoints. While the dosage units (μg/mL and mg/L) are equivalent, the studies investigated distinct endpoints, with the Allium cepa assay focusing on chromosomal aberrations in plant cells and the Neuro-2A study evaluating DNA fragmentation in mammalian neuronal cells.^207,208 Furthermore, in human lung carcinoma cell line A549, where exposure to a copper/CuONPs composite induced DNA fragmentation, as evidenced by increased migration of DNA from the nucleus.²⁰⁹ These findings suggest that composite systems containing both metallic copper and CuONPs can significantly contribute to genotoxicity.²⁰⁹

CuONPs Induced Cell Death Processes

Cell death is a fundamental biological process essential for maintaining tissue homeostasis by eliminating damaged or dysfunctional cells. Depending on the nature and intensity of the stimulus, cells may undergo different forms of death, including apoptosis, autophagy, and cuproptosis.

Apoptosis and autophagy are essential molecular processes that contribute to maintaining organismal and cellular homeostasis, respectively.²¹⁰ Apoptosis facilitates the controlled elimination of damaged or dysfunctional cells, whereas autophagy is involved in the selective degradation and recycling of intracellular components to preserve cellular integrity.²¹⁰ Upon exposure to CuONPs, distinct cell death responses were observed between dog kidney epithelial cell line MDCK and mouse hepatocyte liver cell line AML-12.²¹¹ In MDCK cells, only a small proportion of cells underwent apoptosis, whereas approximately 80% of AML-12 cells exhibited apoptotic cell death.²¹¹ Despite the low levels of apoptosis in MDCK cells, significant cytotoxicity was observed, suggesting the involvement of alternative cell death mechanisms.²¹¹ Further analysis revealed a marked increase in autophagosome formation, indicating that autophagy contributes substantially to cell death in MDCK cells.²¹¹ These findings highlight that CuONPs induced cytotoxicity is highly cell-type dependent.²¹¹ The cytotoxic effects of CuONPs on cancer cell lines are influenced by their conjugation with anticancer agents.²¹² This is demonstrated in studies involving MDA-MB-231 and A549, where treatment with CuONPs conjugated with lapatinib which is an anticancer compound resulted in a marked increase in apoptosis, from 0.65% to 68.96% and from 1.11% to 44.11%, respectively.²¹² These findings suggest that functionalization of CuONPs enhances their apoptotic potential in cancer cells. The method of synthesis of CuONPs can significantly influence their apoptotic potential.²¹² Mitophagy is a selective form of autophagy responsible for the removal of damaged or aged mitochondria, thereby maintaining mitochondrial quality and cellular homeostasis.²¹³ Studies have demonstrated that CuONPs can disrupt this process by blocking autophagic flux and impairing mitophagy, leading to the accumulation of dysfunctional mitochondria.²¹⁴ This accumulation subsequently interferes with mitochondrial energy metabolism and contributes to cellular dysfunction.²¹⁴

In addition to the well-established process of apoptosis, multiple regulated cell death pathways have been increasingly recognized. Several metal ions, including iron, copper, zinc, and Mn, are required in trace amounts for normal physiological functions; however, disturbances in their levels can initiate distinct cell death mechanisms. For example, ferroptosis is a form of cell death associated with iron accumulation and is driven by excessive lipid peroxidation.²¹⁵ In contrast, cuproptosis represents a copper ion dependent mechanism of cell death that arises from mitochondrial dysfunction, characterized by the accumulation of lipoylated mitochondrial proteins and the depletion of iron–sulphur cluster-containing proteins.²¹⁶ Recent studies have demonstrated CuONPs can induce cuproptosis in murine macrophage cell line RAW 264.7.²¹⁷ This process is associated with mitochondrial membrane damage, leading to excessive generation of ROS and disruption of intracellular redox homeostasis. The resulting mitochondrial dysfunction and oxidative imbalance may further contribute to the induction of ferroptosis.²¹⁷ In another study, CuONPs were engineered as nanozymes targeting cysteine (Cys), a key amino acid involved in tumour proliferation.²¹⁸ These nanozymes effectively catalysed the depletion of intracellular Cys, while the released copper ions contributed to the activation of cuproptosis.²¹⁸ Furthermore, Cys depletion has been widely reported to promote ferroptosis in various in vivo models, highlighting a synergistic interplay between these cell death pathways.²¹⁹ A summary of the in vitro pathophysiological effects of CuONPs on different cell lines is presented in Table 1.

Table 1.

Summary of the Cytotoxic, Oxidative Stress, Inflammatory, Genotoxic, Apoptotic, Ferroptotic, and Cuproptotic Effects of Copper Oxide Nanoparticles (CuONPs) of Different Sizes and Morphologies in Various in Vitro Cell and Experimental Models

CuONPs Size	Dose	Experimental Model/Cell Line	Observed Effects	Reference
60 ± 23 nm	0.1, 1, 10, 100 µg/mL (cytotoxicity and functional cardiotoxicity assays); 0.1, 1, 10 µg/mL (gene expression/quantitative polymerase chain reaction assays)	3D bioprinted human iPSC-derived cardiac micro-tissue	Decreased cardiac microtissue viability (1–100 μg/mL) with complete loss of viability and contractility at 100 μg/mL. Increased mitochondrial biogenesis markers and extrinsic apoptosis activation.	[220]
55.80 ± 8.70 nm	5, 10, 20, 30 µg/mL	Human skin epidermal cell line HaCaT	Concentration- and time-dependent cytotoxicity with increased lactate dehydrogenase (LDH) release (10-30 µg/mL), reactive oxygen species (ROS) generation, deoxyribonucleic acid (DNA) damage (peak at 20 µg/mL), and caspase-3 activity (5-20 µg/mL).	[137]
84.68 ± 6.4 nm	2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 25, 30, 35, 40 µg/mL	Canine kidney epithelial cell line MDCK and murine hepatocyte cell line AML-12	MDCK: Concentration-dependent cytotoxicity with half maximal inhibitory concentration (IC50) = 21.9 μg/mL. Apoptosis (<30%) and autophagy-dependent cell death with increased autophagosome formation (8–30 μg/mL). ROS decreased up to 14 μg/mL and increased at 16–20 μg/mL. Dose-dependent Gap 2/mitosis (G2/M) cell cycle arrest (>16 μg/mL). AML-12: Concentration-dependent cytotoxicity (IC50 = 17.6 μg/mL). Early apoptosis (approximately 80% cell death). ROS decreased up to 14 μg/mL and increased at 16–20 μg/mL. Dose-dependent G2/M cell cycle arrest (>16 μg/mL).	[211]
<50 nm	5, 10, 50, 100, 200, 400 µg/mL (for cytotoxicity assay); 5, 25, 50 µg/mL (for Endoplasmic reticulum (ER) stress and morphology)	Human cerebral microvascular endothelial cell line hCMEC/D3	Decreased cell viability (≥10 μg/mL; lowest at 400 μg/mL). Dose- and time-dependent increases in apoptosis. Dose and time dependent caspase-3/7 mediated apoptosis (highest at 200 μg/mL). Morphological alterations with ER vacuolization (5–25 μg/mL).	[221]
25–50 nm	1, 10, 50, 100,200 µg/mL (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and apoptosis assays); 1, 10, 20, 50, 100, 200 µg/mL (LDH assay)	Peripheral blood mononuclear cells	MTT assay showed dose-dependent decreases in cell viability (no effect at 1 μg/mL). Dose-dependent increases in LDH release from 1 μg/mL. Apoptosis with nuclear condensation at 47.5 μg/mL (IC50).	[222]
73.9 ± 20.7 nm (length) and 14.7 ± 3.9 nm (width) copper oxide (CuO) nanotubes	25, 35, 50,100, 200mg/L	Tetrahymena thermophila (free-living freshwater ciliate protozoan)	Dose- and time-dependent decreases in cell viability (median lethal concentration = 82.43 mg/L). Early increase in superoxide radicals and altered ROS profile. Increased autophagy and autophagosome formation (demonstrated at 35 mg/L).	[223]
43.1 and 49.5 nm	5, 10, 20, 30, 40, 50, 60 μg/mL (MTT assay); 5, 10, 20 μg/mL (intracellular copper measurement); other mechanistic experiments used 20 μg/mL	Murine alveolar macrophage cell line MH-S	Dose-dependent decreases in cell viability and increases in intracellular copper levels. Increased ferredoxin 1 (FDX1), dihydrolipoamide S-acetyltransferase (DLAT), dihydrolipoamide S-succinyltransferase (DLST), copper transporter 1 (CTR1), and significant increase in interleukin (IL)-17A expression . Cell death reached 23.6% at 20 μg/mL.	[30]
80 nm	5, 10,20 µg/mL	Murine macrophage cell line RAW264.7	Increased intracellular copper levels. Dose-dependent upregulation of DLAT, lipoic acid synthetase, FDX1, and heat shock protein 70 (HSP70). Increased transferrin receptor, ferroportin, ferritin heavy chain, malondialdehyde (MDA), and lipid peroxidation levels.	[217]
14.21 ± 0.94 nm of (3-carboxypropyl) triphenylphosphoniumbromide-derived hyaluronic acid-modified CuO nanorods	4, 6, 8, 10, 20,40 μg/mL (cell inhibition assay); 5, 10, 15 μg/mL (cysteine [Cys] and glutathione [GSH] depletion assays); 15 μg/mL (ferroptosis and cuproptosis assays)	Human breast adenocarcinoma cell line MCF-7	Dose-dependent inhibition of cell viability. Increased ROS generation. Catalytic depletion of Cys and GSH (35.2% and 46.7% of control levels, respectively at 15 μg/mL). Ferroptosis characterized by reduced mitochondrial size, loss of mitochondrial cristae, and decreased glutathione peroxidase 4 expression. Cuproptosis characterized by DLAT aggregation and loss of iron-sulphur cluster- proteins.	[218]
112 ± 16 nm of elesclomol-loaded CuONPs	0.4 μg/mL	Murine melanoma cell line B16	Increased cell membrane injury, necrosis (1.9-fold vs positive control), DLAT foci aggregation, and LDH release.	[143]
19.94 ± 3.80 nm (copper/CuONPs)	Human lung normal cell line WI-38: 5–10,000 μg/mL (MTT assay); 201.26 μg/mL (Comet assay and oxidative stress parameters) Human lung adenocarcinoma epithelial cell line A549: 5–10,000 μg/mL (MTT assay); 209.70 μg/mL (Comet assay and oxidative stress parameters)	WI-38 and A549	WI-38: Strong inhibition of cell growth. DNA damage at IC50. Increased nitric oxide (NO), with decreased GSH, MDA, and hydrogen peroxide (H2O2) levels. A549: Strong inhibition of cell growth. DNA damage at IC50. Decreased NO and H2O2 levels, with increased superoxide dismutase activity.	[209]
70 – 100 nm	6.25, 12.5, 25, 50, 100, 200 and 400 mg/L (cell viability assay); 12.5, 25, 50 and 100 mg/L (MDA, DNA fragmentation and micronucleus assays)	Murine neuroblastoma cell line Neuro- 2A	Increased mitochondrial activity at lower concentrations and decreased mitochondrial activity at higher concentrations. Increased MDA at 25 mg/L. Increased DNA fragmentation up to 50 mg/L, followed by reduced DNA fragmentation at 100 mg/L. Increased micronucleus frequency at 12.5 mg/L.	[208]
52.51 ± 10.23 nm	10, 25, 50 µg/mL	A549	Dose-dependent decreases in cell viability. Increased MDA, decreased GSH, and increased HSP70, p53, and Rad51 recombinase protein expression.	[224]
34.9 nm cupric oxide nanoparticles	2.5, 5, 10, 15, 20, 40 µg/mL (cytotoxicity analysis); 5, 10, 15, 20 µg/mL (genotoxicity assay); 7.5, 12.5, 20 µg/mL (apoptosis assay in HepG2) and 5, 10, 20 µg/mL (apoptosis assay in Caco-2); 5, 10, 15, 25 µg/mL (oxidative damage assay)	Human hepatocellular carcinoma cell line HepG2 and Human colorectal adenocarcinoma cell line Caco-2	HepG2: Dose-dependent reduction in cellular metabolic activity and cell disruption. DNA damage. Increased MDA and decreased GSH with apoptosis. Caco-2: Dose-dependent reduction in cellular metabolic activity and cell disruption. Greater DNA damage than HepG2 cells. Increased MDA (significant at ≥10 µg/mL) and decreased GSH with apoptosis and higher necrosis.	[225]
12 nm (rod shaped CuONPs) and 50 nm (spherical CuONPs)	5-100 µg/mL (cell viability assay) ; 5, 10, 25, 50 µg/mL (oxidative stress); 25 µg/mL for pro inflammatory effects	HepG2	Dose-dependent decreases in cell viability, with greater toxicity of rod-shaped CuONPs. Dose-dependent increases in ROS production. Overexpression of chemokines, interleukins (IL-18, IL-12A, IL-8, and IL-7), and tumour growth factor-β1.	[31]
< 50 nm	3, 10, 30 cm2/mL	A549	Increased cytotoxicity, IL-8 expression, pro-inflammatory transcription factor activation, and nuclear factor kappa B activation at low doses. Decreased pro-inflammatory transcription factor activation at higher doses.	[226]
< 50 nm (commercial CuONPs), 160 nm (sonochemical CuONPs prepared in ethanol) and 70 nm (sonochemical CuONPs prepared in water)	10, 50, 100 µg/mL	Caco-2 (analysis is done on the apical [Ap] and basolateral [BI] compartments)	Commercial CuONPs: Increased IL-6 release at 100 µg/mL and IL-8 release at all BI exposure concentrations. Sonochemical CuONPs: Increased IL-8 release after Ap (100 µg/mL) and BI exposure (all concentrations).	[203]
< 50 nm	0.25, 0.5, 1, 2 µg/mL	Human airway epithelial cell line NCI- H292	Dose-dependent increases in IL-6, IL-8, and extracellular signal-regulated kinase phosphorylation.	[227]

In vivo Toxicity of CuONPs

In this section, the in vivo toxicity effects of CuONPs are reported according to the major routes of exposure such as inhalational, i.t., oral and parenteral routes.

Inhalation and Intratracheal Exposure

Following nose-only inhalation exposure to CuONPs for 4 h/day, 5 days/week over a 2-week period, CuONPs were found to modulate pulmonary immune responses in a manner dependent on the pre-existing immune status of the mice.¹²¹ In healthy mice, CuONPs exposure reduced pulmonary type 1 helper T cells and type 2 helper T cells (T_H2) cell populations while increasing T-bet⁺ regulatory T (Treg) cells.¹²¹ Similarly, in asthmatic mice, CuONPs exposure suppressed T_H2-mediated immune responses, as evidenced by reduced T_H2 cell numbers and lower IL-4 levels, accompanied by increased T-bet⁺ Treg cells.¹²¹ In contrast, CuONPs exposure in allergen immunotherapy treated asthmatic mice enhanced T_H2 responses, resulting in increased T_H2 and Treg cell numbers.¹²¹ In another study, 6 weeks of CuONPs exposure in mice promoted adaptive immune activation, as shown by increased T-cell proliferative responses and elevated production of both type 1 helper T lymphocytes and T_H2 associated cytokines.³⁵ In contrast, innate immune function was impaired, evidenced by reduced granulocyte phagocytic activity, indicating a decreased capacity of these cells to perform normal first-line defence functions.³⁵ A major strength of this study is the use of a sub-chronic inhalation exposure model for 6 weeks, which more closely reflects potential occupational or environmental exposure to airborne CuONPs.³⁵ The i.t. administration of CuONPs in C57BL/6 mice resulted in significant accumulation of copper ions in lung tissue, accompanied by pulmonary inflammation characterized by inflammatory cell infiltration, increased alveolar wall thickness, and disruption of alveolar architecture.³⁰ Haematological alterations, including elevated white blood cells (WBC) and monocyte counts, further indicated activation of systemic inflammatory responses.³⁰ Mechanistically, the study suggested that CuONPs induced pulmonary toxicity may involve FDX1-mediated cuproptosis and IL-17A associated inflammatory signalling pathways.³⁰ However, the study evaluated only short-term exposure (7 days), which may not fully reflect chronic occupational or environmental exposure scenarios.³⁰ Furthermore, another study investigated the combined pulmonary effects of different nanoparticles following i.t. instillation into female rat lungs and found that CuONPs exhibited the highest pulmonary toxicity among the tested nanoparticles.²²⁸ Combined exposure of CuONPs and nickel oxide nanoparticles produced an additive increase in lung inflammation, whereas co-exposure with carbon black nanoparticles exerted an antagonistic effect by reducing inflammatory responses.²²⁸ These findings suggest that the toxicity of CuONPs can be modulated by co-exposure with specific nanoparticles, highlighting the importance of nanoparticle interactions in nanotoxicological risk assessment.²²⁸ In a different study, i.t. instillation of CuONPs revealed dose-dependent elevation in the inflammatory and the cellular markers in BALF.³⁴ CuONPs exposure altered antioxidant enzyme expression, with decreased CAT, glutathione peroxidase-1, and peroxiredoxin-2 levels and increased SOD-2 expression.³⁴ Interestingly, the compensatory increase in SOD-2 expression may reflect an adaptive cellular response to excessive ROS generation, whereas depletion of other antioxidant enzymes indicates impaired antioxidant defence capacity.³⁴ Conversely, short-term inhalation exposure to CuONPs for 5 days in rats induced pulmonary inflammation and increased expression of the proliferation-associated protein “epithelial cell transforming sequence 2” within bronchoalveolar epithelial cells, suggesting enhanced epithelial cell proliferation following exposure.²²⁹ Neonatal inhalation exposure to CuONPs in C57BL/6J mice predominantly affected the male striatum, causing increased dopaminergic activity, reduced gamma-aminobutyric acid inhibition, impaired serotonergic signalling, and disruption of excitatory–inhibitory neurotransmitter balance.²³⁰ Similarly, i.t. instillation of CuONPs in BALB/c mice exhibited greater pulmonary cytotoxicity, as evidenced by increased LDH release and oxidative damage.²³¹ Furthermore, CuONPs exposure induced epigenetic alterations characterized by increased DNA methylation and disruption of DNA methylation regulatory machinery.²²³ Dysregulation of transposable elements was also observed, suggesting the potential for genomic instability.²³¹ Notably, in a separate study using a similar murine model, i.t. instillation of CuONPs induced vascular toxicity, characterized by shortened thrombotic occlusion times in both arterioles and venules, together with systemic oxidative stress and inflammation.¹¹⁹ These findings further demonstrate the prothrombotic potential of pulmonary CuONPs exposure and its ability to disrupt vascular homeostasis.¹¹⁹

Oral Exposure

The oral route represents a critical pathway for CuONPs induced toxicity. This route closely mimics the primary means of human exposure, occurring through ingestion of contaminated food, water or via swallowing of inhaled particles cleared from the respiratory tract.²³² Oral gavage administration of CuONPs (5 and 50 mg/kg body weight/day) in Wistar rats resulted in decreased hepatic GSH, CAT, and SOD activities, accompanied by elevated MDA levels, indicating disruption of the antioxidant defence system and enhanced lipid peroxidation.²³³ These findings suggest that oxidative stress is a major contributor to CuONPs induced hepatotoxicity following oral exposure.²³³ CuONPs, ZnONPs, and their combined exposure were administered at doses of 1, 5, and 25 mg/kg/day for 14 days in mice.²³⁴ The results demonstrated dose-dependent oxidative stress in the liver and kidney tissues of mice, as evidenced by reduced SOD and CAT activities and decreased GSH levels, accompanied by increased GST activity.²³⁴ Elevated thiobarbituric acid reactive substances (TBARS) levels indicated enhanced lipid peroxidation, while increased heat shock protein 70 and 8-hydroxy-2′-deoxyguanosine (8-OHdG) levels reflected cellular stress and oxidative DNA damage.²³⁴ Notably, co-exposure to CuONPs and ZnONPs produced the most pronounced alterations in all biomarkers, suggesting a synergistic toxic effect.²³⁴ Bugata et al²³⁵ further evaluated the acute and subacute oral toxicity of CuONPs in female Wistar rats as per the Organisation for Economic Co-operation and Development guidelines which provide internationally standardized procedures for evaluating acute oral toxicity and repeated-dose 28-day oral toxicity in rodents, thereby ensuring the reliability and reproducibility of toxicity assessments.^236,237 A single oral gavage dose of 2000 mg/kg and repeated oral administration of 30, 300, and 1000 mg/kg/day for 28 days in rats did not cause mortality or marked changes in body weight, feed intake, or most haematological parameters.²³⁵ However, higher CuONPs doses (2000 mg/kg in the acute study and 1000 mg/kg/day in the subacute study) produced significant biochemical and oxidative stress alterations in the liver, kidney, and brain, including increased MDA levels, reduced GSH content, altered SOD and CAT activities, elevated hepatic and renal transaminase activities, and reduced brain acetylcholinesterase activity.²³⁵ It was also identified that the potential toxicity is due to the generation of ROS.²³⁵ In another study, haematological assessment following gastric gavage administration of CuONPs in rats for 28 days demonstrated significant reductions in red blood cells, haemoglobin, hematocrit, mean corpuscular volume, and platelet counts along with elevated WBC levels.²³⁸ These alterations suggest that CuONPs may induce hematotoxicity characterized by anaemia-like conditions. However, the study employed relatively high exposure doses (300 mg/kg/day), which may limit direct extrapolation to realistic human exposure scenarios.²³⁸ Similarly, the cognitive effects of CuONPs were evaluated using behavioural testing.²³⁹ In the open-field locomotor activity test, CuONPs did not significantly alter normal locomotion or exploratory behaviour in rats. However, elevated plus maze analysis demonstrated increased anxiety-like behaviour following CuONPs exposure.²³⁹ In the Morris water maze, no marked impairment in spatial learning or memory retention was observed, although transient alterations in escape latency were reported during certain training sessions.²³⁹ Collectively, these findings suggest that short-term oral exposure to CuONPs may exert a greater effect on emotional behaviour than on the cognitive or locomotor functions.²³⁹ Consistent with these findings, the authors also reported a similar oxidative stress profile in brain tissue of Swiss albino mice, characterized by decreased GSH and increased GST levels and increased TBARS and 8-OHdG levels, impairment of the brain antioxidant defence system, accompanied by oxidative damage to key biomolecules, including DNA, lipids, and proteins.²⁴⁰ In another comparative study evaluating the reproductive toxicity of CuONPs and copper sulphate (CuSO₄) in mice, oral gavage exposure to equivalent doses of both copper forms resulted in significant reductions in body weight and reproductive organ weights, indicating adverse effects on the male reproductive system.²⁴¹ Furthermore, both treatments negatively affected fertility outcomes; however, CuSO₄ induced more pronounced pathological alterations than CuONPs.²⁴¹ These findings suggest that the ionic form of copper may possess greater toxic potential than its nanoparticulate counterpart under equivalent exposure conditions.²⁴¹ Similarly, female mice were orally exposed to CuONPs at doses of 1.25–5 mg/kg/day for 30 days to evaluate their effects on ovarian function and oocyte maturation.²⁴² CuONPs exposure significantly reduced oocyte developmental competence and impaired meiotic progression in a dose-dependent manner.²⁴² Moreover, CuONPs treated oocytes exhibited abnormal spindle assembly along with chromosome misalignment. A marked reduction in α-tubulin acetylation was also observed, indicating decreased microtubule stability and disrupted microtubule dynamics.²⁴² These findings suggest that CuONPs adversely affect oocyte maturation by interfering with cytoskeletal organization and meiotic spindle integrity.²⁴²

Parenteral Exposure

The parenteral route of administration bypasses the gastrointestinal tract and delivers substances directly into systemic circulation,²⁴³ thereby enabling controlled evaluation of nanoparticle-induced toxicity across various organs. In an in vivo study conducted in adult male Wistar rats, administration of small-sized CuONPs (25 nm) at a dose of 2 mg/kg over 7 weeks resulted in marked ultrastructural alterations in hepatic tissue, with mitochondria identified as a primary target.²⁴⁴ These alterations included mitochondrial swelling, cristae disruption, and matrix lysis, indicating impaired mitochondrial integrity. In addition to mitochondrial damage, broader hepatocellular toxicity was also observed.²⁴⁴ The molecular mechanisms underlying this mitochondrial dysfunction were not fully elucidated in the study.²⁴⁴ However, given the central role of mitochondria in ATP production via the tricarboxylic acid (TCA) cycle, it is reasonable to suggest that such structural damage may disrupt cellular energy metabolism.²¹⁴ Supporting this hypothesis, it was identified that CuONPs can lead to mitochondrial dysfunction which can lead to reduced TCA activity.²¹⁴ While these findings suggest a potential link between mitochondrial damage and metabolic dysfunction, direct causal relationships remain to be established.²¹⁴ Furthermore, CuONPs exposure in male rats has been associated with significant increase in hepatic injury markers, including alanine aminotransferase, aspartate aminotransferase, and MDA, reflecting hepatocellular damage and oxidative stress.²⁴⁵ Notably, such effects were observed within 4 weeks at a higher dose of 200 mg/kg.²⁴⁵ However, the elevated dose may limit physiological relevance and potentially exaggerate the observed toxic effects compared to real-world exposure levels.²⁴⁵ Furthermore, the mechanisms underlying these effects remain insufficiently characterized in in vivo studies. Supporting mechanistic insights from in vitro studies indicate that CuONPs can induce ROS generation, DNA damage, and apoptosis in epithelial kidney cell line from Xenopus laevis A6.²⁴⁶ It is noteworthy that some studies report greater toxicity with larger nanoparticles, which contrasts with the commonly observed size-dependent increase in toxicity for smaller particles.^246,247 The i.p. administration of chemically and green synthesized CuONPs in / BALB/c mice elevated LDH, SGOT, and creatinine levels, indicating hepatic and renal dysfunction.¹⁴⁰ Chemically synthesized CuONPs induced stronger inflammatory responses and more severe histopathological damage in the liver and kidney compared to green synthesized CuONPs.¹⁴⁰ These findings suggest that the synthesis route significantly influences the toxicity profile of CuONPs.¹⁴⁰ In neuronal models, CuONPs-induced neurotoxicity has been shown to depend on both particle size and surface modification. Smaller uncoated CuONPs exhibited greater inhibition of acetylcholinesterase activity than the larger uncoated CuONPs, with smaller particles also inducing greater expression of the neurodegeneration associated gene, potentially linked to increased copper ion release.²⁴⁸ Overall, these findings suggest that CuONPs toxicity is influenced by the combination of multiple factors, including particle size, surface modification, and ion release, rather than size alone.²⁴⁸ A summary of the in vivo toxicity profiles of CuONPs following inhalational, oral, and parenteral exposures is presented in Table 2.

Table 2.

Overview of Reported in vivo Toxicological Outcomes Following Copper Oxide Nanoparticles (CuONPs) Exposure in Experimental Animal Models, Including Oxidative Stress, Inflammation, Histopathological Alterations, Organ Toxicity, and Cell Death-Related Mechanisms

Size	Dose	Model	End-Point Measurement	Effects	References
21 ± 4 nm	0.45 mg/kg	Albino female rats	Intranasal administration 3 times/week for 6 weeks; behavioural assessment throughout; sacrificed after 6 weeks	Decreased hematocrit. Increased reticulocytes, platelet count, proportion of axons with damaged myelin sheath and vascular mitochondrial morphotypes.	[249]
< 50 nm	5, 10, 25 mg/kg	Male Wistar rats	Intraperitoneal (i.p.) injection for 9 days; sacrificed on day 9	Mild to moderate hepatic necrosis, hepatic ray disorganization, and moderate nuclear/cytoplasmic caspase-3 immunoreactivity at 5–10 mg/kg. Severe diffuse hepatocyte necrosis, complete hepatic cord disorganization, and diffuse hepatic caspase-3 immunolocalization at 25 mg/kg.	[32]
43.96±12.36 nm	Normal mice (toxicity study) (0.1, 0.2, 0.4 and 1 mg/kg); Chronic obstructive pulmonary disease (COPD) + CuONPs group (0.25, 0.5, 1.0 mg/kg); Thioredoxin-interacting protein (TXNIP) overexpressed group (0.5 mg/kg); TXNIP knock out model (0.5 mg/kg)	C57BL/6 male mice	Intranasal administration (3 doses). Normal mice group: days 1, 3 and 5; sacrificed on day 7. COPD, TXNIP overexpression and TXNIP knockout groups: days 8, 10 and 12; sacrificed on day 15	Effect of CuONPs on normal mice: CuONPs (>0.2 mg/kg) increased BALF inflammatory cell counts, including neutrophils, macrophages, and lymphocytes. Increased inflammatory cytokines such as interleukin (IL)-1β, IL-6, and tumour necrosis factor alpha (TNF-α), inflammatory cell infiltration, and mucus production were also observed. Effect of CuONPs on COPD model: Increased inflammatory cell counts and cytokine levels. Aggravated COPD pathology with increased inflammatory cell infiltration, mucus secretion, oxidative stress (8-hydroxy-2′-deoxyguanosine and malondialdehyde [MDA]), and decreased superoxide dismutase (SOD) activity. Effect of TXNIP overexpression: Further aggravated CuONPs induced COPD pathology with increased inflammatory cell infiltration, cytokine production, and oxidative stress. Effect of TXNIP knockout: Attenuated CuONPs induced inflammation and COPD exacerbation with reduced inflammatory cell counts cytokine production, oxidative stress, and mucus secretion.	[250]
51 nm	5, 10, 20, 40, 60, 80, 100 and 120 ppm (median lethal concentration assay); 40 and 60 ppm (sub-lethal assay)	Danio rerio (zebrafish embryo)	Continuous immersion in CuONP-containing medium; endpoints assessed up to 96 hours (h) post-fertilization	Concentration-dependent heartbeat retardation, hatching delay, and reactive oxygen species generation.	[251]
50 – 100 nm	10 and 25 mg/kg	C57BL/6 male mice	Daily treatment for 2 weeks, the sacrifice was performed 12–16 h after the last gavage	Decreased body weight, testicular weight, and testicular organ coefficient in all exposure groups. Increased injured seminiferous tubules at 25 mg/kg and reduced Leydig cell number across all groups.	[252]
12 ± 1 nm of copper nanoparticles	3.68 mg/m3 copper nanoparticles	C57BL/6 male mice	Inhalation exposure (4 h/day, 5 days/week) for 2 weeks; necropsied within 1 h or 3 weeks after exposure	0 week: Neutrophilia; increased bronchoalveolar lavage fluid (BALF) total cells, macrophages, lymphocytes, total protein, lactate dehydrogenase (LDH), increased inflammatory cytokines, perivasculitis and alveolitis. 3 week: Persistent increase in BALF inflammatory cells; BALF protein, LDH, cytokines, and lung histopathology returned to baseline.	[114]
10 nm	0.6, 2.4, 3.3, 6.3 and 13.2 mg/m3	Adult male outbred rats (HsdCpb: WU)	Inhalation exposure (18 min–6 h/day) for 5 consecutive days; sacrificed on day 6 or day 28 after recovery	Day 6: Increased total BALF cells, macrophages, neutrophils, lymphocytes, LDH, alkaline phosphatase (ALP), total protein, N-Acetyl-β-D-glycosaminidase (NAG), alveolitis, bronchiolitis, vacuolation of respiratory epithelium, emphysema, and olfactory epithelial degeneration. Day 28: Persistent increase in total BALF cells and macrophages; limited residual lung inflammation at the highest dose; neutrophils, lymphocytes, LDH, ALP, total protein, and NAG returned to baseline/no treatment-related effect.	[115]
40 nm	6.2–7.1 mg/kg	C57BL/6J male mice	Single intratracheal (i.t.) instillation; sacrificed on day 7	Increased white blood cell (WBC) and monocyte/WBC ratio. Pulmonary inflammation with alveolar wall thickening and alveolar structural disruption. Increased expression of cuproptosis-related genes (dihydrolipoamide S-acetyltransferase, dihydrolipoamide S-succinyltransferase and copper transporter 1).	[30]
33 nm	2 mg/rat	Male F344/DuCrlCrj rats	Single i.t. instillation; sacrificed on days 1 and 28	Day 1: Severe lung oedema. Increased 5-bromo-2′-deoxyuridine-positive and inducible nitric oxide synthase-positive cells in the lungs. Day 28: No data due to mortality.	[253]
< 50 nm	1 and 5 mg/kg	Adult male Wistar albino rats	Single i.t. instillation; sacrificed after 24 h or 1 week	Increased LDH, ALP, total protein, and total leukocytes; decreased catalase (CAT) and SOD activity; increased degeneration, fibrosis, and granuloma formation (more severe at 1 week).	[116]
55 nm	0.15 and 1.5 mg/kg	Male Sprague Dawley rats	Single i.t. instillation; sacrificed after 24 h	Increased BALF total cell counts, polymorphonuclear neutrophils (PMN), LDH activity, and total protein. Decreased BALF macrophages. Acute suppurative inflammation around the terminal bronchioles and alveoli, with pulmonary oedema at 1.5 mg/kg.	[34]
23.1 nm	16, 24 and 48 cm2/rat	Female Wistar rats	Single i.t. instillation; sacrificed after 24 h	Dose-dependent increases in BALF neutrophils, LDH, total protein, monocyte chemoattractant protein-1, and cytokine-induced neutrophil chemoattractant-3.	[228]
<50 nm	30, 50 and 100 μg/animal	C57BL/6J male mice	Single i.t. instillation; sacrificed on day 3	Wrinkled and deformed nuclei with chromatin condensation in the cerebral cortex. Reduced Nissl bodies. Increased IL-6 and tumour necrosis factor mRNA expression.	[254]
50 nm	1.5, 2.5 and 5 mg/kg	Wild-type C57BL/6J male mice and Microtubule-associated protein 1 light chain 3 beta (lc3b−/−) knockout mice.	Single i.t. instillation and after 3 days the mice were sacrificed	Wild-Type (C57BL/6J) mice: Dose-dependent lung injury with inflammatory cell infiltration, alveolar wall thickening, and alveolar structural disruption. Increased IL-6, IL-1β, TNF-α, heme oxygenase-1, and glutamate-cysteine ligase modifier subunit expression. lc3b−/− mice: More severe lung injury, increased BALF cell counts and protein concentration, and elevated IL-6 and IL-1β expression.	[255]
15 – 20 nm	1, 2, 4, 8, 16, 32, 64 and 512 mg/kg	Male-specific pathogen-free rats (RjHan: WI)	Oral gavage once daily for 5 days; sacrificed on day 6 or day 26	At day 6, doses ≤32 mg/kg showed no mortality or signs of toxicity, whereas 512 mg/kg caused slight weight loss. Decreased WBC at 32–64 mg/kg and a two-fold increase at 512 mg/kg. Reduced red blood cell (RBC) parameters and increased LDH at 64 mg/kg. At day 26, WBC and RBC parameters returned to control levels at doses ≤ 32 mg/kg. Increased gastric inflammation and Kupffer cell hypertrophy.	[33]
< 50 nm	5 and 50 mg/kg	Male Wistar albino rats	Oral gavage daily for 14 days; sacrificed after day 14	Dose-dependent decreases in reduced glutathione, CAT, and SOD, with increased MDA levels.	[233]
< 50 nm	150 cm2/rat	Female Wistar rats	Single i.t. instillation; sacrificed after 24 h or 4 weeks	Effects observed at 24 h: Increased total BALF cells, PMNs, eosinophils, BALF LDH, total protein, IL-1β, and macrophage inflammatory protein-2. Effects observed after 4 weeks: No persistent inflammatory response; BALF LDH, total protein, and IL-1β returned to control levels (normalized).	[226]
20 ± 5 nm	2 mg/kg	Male Wistar albino rats	Daily i.p. injection (5 injections/week) for 7 weeks; sacrificed 24 h after the last injection	Morphometric alterations, including back arching and abdominal ballooning. Hepatic histological alterations, including Kupffer cell hyperplasia, sinusoidal dilatation, hepatocytes anisokaryosis and inflammatory cells inflammation. Hepatocyte swelling, mitochondrial crystolysis and matrix lysis, with chromatin clumping, euchromatin fragmentation, and micronucleus formation.	[244]

Conclusions and Future Perspectives

This review provides a comprehensive overview of CuONPs, focusing on their synthesis, physicochemical characteristics, biodistribution, pharmacological applications, and associated pathophysiological effects. Current evidence suggests that the biological behaviour of CuONPs is strongly influenced by properties such as particle size, morphology, surface charge, dissolution behaviour, and surface functionalization. These factors play a critical role in determining cellular uptake, biodistribution, protein corona formation, therapeutic performance, and toxicity. CuONPs exhibit considerable biomedical potential, particularly due to their broad-spectrum antibacterial activity, anticancer properties, antidiabetic effects, and antioxidant capabilities. Their therapeutic effects are largely mediated through ROS generation, copper ion release, and modulation of cellular signalling pathways. However, these same mechanisms are also responsible for many of their toxicological effects, including oxidative stress, inflammation, mitochondrial dysfunction, DNA damage, and activation of regulated cell death pathways such as apoptosis, autophagy, ferroptosis, and cuproptosis. In vivo studies further demonstrate that CuONPs may induce organ-specific toxicities involving the lungs, liver, kidneys, brain, reproductive organs, and haematological systems depending on exposure route, dose, exposure duration, and nanoparticle properties. A major challenge emerging from the current literature is the dual nature of CuONPs as both promising nanotherapeutic agents and potential toxicological hazards. This highlights the need to carefully balance biological efficacy with safety during the design for the development of CuONPs based nanomedicines. Despite significant progress in preclinical research, the clinical translation of CuONPs remains restricted by several important limitations, including insufficient long-term toxicity data, incomplete understanding of biodistribution and clearance, lack of standardized dosing approaches and exposure thresholds, manufacturing reproducibility challenges, and the absence of harmonized regulatory frameworks for nanomedicines.

Although substantial evidence underscores both the therapeutic potential and toxicological risks of CuONPs, notable knowledge gaps remain. Future studies should focus on establishing standardized approaches for nanoparticle characterization and biological evaluation to improve comparability across studies and facilitate clinical translation. Systematic investigations are also required to determine both local and systemic accumulation patterns following different exposure routes, enabling a clearer understanding of tissue-specific toxicity and long-term biodistribution. Furthermore, long-term studies in higher mammalian models are needed to better assess chronic health effects and real-world safety profiles. Toxicological evaluations should also be extended to disease-relevant animal models, including asthma, hypertension, obesity, and diabetes, as these pre-existing conditions may alter nanoparticle biodistribution and exacerbate toxic responses.

Author Contributions

All authors made a significant contribution to the work reported, whether that is in the conception, study design, execution, acquisition of data, analysis and interpretation, or in all these areas; took part in drafting, revising or critically reviewing the article; gave final approval of the version to be published; have agreed on the journal to which the article has been submitted; and agree to be accountable for all aspects of the work.

Disclosure

The authors report no conflicts of interest in this work.