Potential application and prospects of ROS-sensitive biomaterials in cancer therapy: a immune microenvironment triggered nanomaterial

Abstract

Reactive Oxygen Species (ROS) is the collective term used for the extremely reactive molecules that are important mediators in physiological processes as well as the development of various disease conditions. Normal cells maintain a delicate equilibrium, known as redox homeostasis, between antioxidants and ROS levels. Any imbalance in the redox homeostasis of the body results in oxidative stress which can result in inflammation, necrosis, apoptosis, cell death, and eventually a disease state. Enhanced ROS levels are a key feature in cancer cells that is being explored for developing reactive oxygen species-sensitive biomaterials. The distinct variation in redox potential between normal cells and tumour cells is one of the major physiological differences between them, that has enabled the development of ROS-sensitive nanomaterials for cancer therapy. ROS-sensitive nanomaterials are sensitive to the physiological variations in the cells, like high levels of hydrogen peroxide and glutathione in the cancer cells. ROS-responsive nanomaterials have the unique property of modulating microenvironmental redox conditions in cancer cells. ROS-sensitive material can work either by scavenging the ROS or by simulating the cellular antioxidants, leading to cancer cell cytotoxicity. These ROS-sensitive nanomaterials can simulate the human body’s natural antioxidants like, superoxide dismutase and peroxidase. Thus, ROS-sensitive nanomaterials hold promise as a potential platform for the treatment of cancer. The present review will cover the importance of ROS in cancer, the different types of ROS-sensitive nanomaterials available and their therapeutic application in cancer therapy.

Introduction

Cancer accounts for almost 10 million deaths annually and is the second most recurrent cause of mortality after cardiovascular disorders [1]. Despite the availability of various treatment modalities, one in six cancer patients succumb to it [2]. Poor treatment efficacy is the primary reason for such a high rate of mortality among cancer patients. Chemotherapy is the most frequently employed clinical approach in cancer, however, the lack of cell-specific drug targeting leads to a large number of unwanted toxic side effects and multi-drug resistance. This makes chemotherapy an inefficient treatment modality. Vomiting, nausea, alopecia, nephrotoxicity, cardiomyopathy and mucositis are some of the major side effects in patients undergoing cancer chemotherapy that affect their quality of life badly [1, 3]. The detrimental effect of cancer chemotherapy on the physical and mental health of the patient necessitates the development of an efficicacious drug delivery system with minimum toxic effects. The primary requisite for the novel anticancer drug delivery system includes cell-specific drug targeting, reduced side effects and drug resistance [3].

Smart nanoparticles are garnering significant interest in cancer drug delivery due to their capacity to selectively target specific cells and adapt to various treatment methods. They can be triggered or adjusted to react to particular stimuli, enabling precise, site-specific drug delivery. Additionally, these smart nanoparticles are also capable of co-delivering a drug along with a diagnostic agent resulting in the development of theranostics along with anti-cancer drug delivery [1]. Apart from this, smart nanoparticles have inherent advantages of nanoparticles like large surface area, prolonged circulation, controlled drug delivery and improved permeation across biological barriers [4]. Smart nanomaterials are capable of responding to different endogenous and exogenous stimuli like pH, temperature, magnetic field, electrical energy, enzymes, light, reactive oxygen species (ROS), tumour cell-specific factors etc. [3].

Amongst them, ROS-sensitive materials are being explored extensively for targeting cancer cells. ROS is a collective term used for a series of highly reactive and unstable molecules especially of molecular oxygen species, namely, superoxide anion (O₂∙⁻), hydrogen peroxide (H₂O₂), singlet oxygen (¹O₂), hypochlorous acid (HOCl), nitric oxide (NO∙), thiol peroxyl radicals (RSOO∙), hydroxyl radical (∙OH). ROS are important mediators in physiological processes. Depending upon the site, level and nature, ROS can exert different effects. The ROS at low levels mediates various physiological processes like cellular proliferation, ageing, antimicrobial activity, DNA mutagenesis, protein activation and gene transcription [4]. However, elevated levels of ROS result in lipid peroxidation and damage to DNA and proteins. This condition is known as as oxidative stress which leads to the development of pathophysiological states like inflammation, cardiovascular diseases, neurodegenerative diseases, diseases and cancer [5]. An increase in ROS beyond the acceptable threshold is cytotoxic to the cell thus it is pertinent to maintain the levels of ROS below the cytotoxic threshold. In normal cells, the ROS levels are controlled below cytotoxic levels with the help of intracellular antioxidant enzymes (like Superoxide dismutase SOD family that neutralize, superoxide anion to hydrogen peroxide (H₂O₂), Catalase (CAT) change hydrogen peroxide (H₂O₂) to molecular oxygen (O₂) etc.) that scavenge the excess ROS, thus maintaining redox homeostasis of the cell [6]. An imbalance in the production of ROS and its removal by antioxidants results in elevated levels of ROS and a prooxidant stage that results in pathophysiological disorder. Thus, ROS is a paradoxical situation wherein a perfect and tight balance is a prerequisite for maintaining the general homeostasis of the cell and body [7].

Site of reactive oxygen species (ROS) production

In living organisms, the ROS can be endogenous or exogenous. Mitochondria and cytoplasm form the endogenous source of the ROS in a living organism. Mitochondria, a cellular organelle, is the primary site for cellular energy or ATP generation via, oxidative phosphorylation, wherein, oxygen is utilized to produce ATP molecules. However, inefficient oxidative phosphorylation in the mitochondria can result in the generation of ROS. In the mitochondrial electron transport chain, electrons escape the electron transport chain to combine directly with the molecular oxygen (O₂) to form superoxide anions (O₂⁻). Further, manganese or copper/zinc superoxide dismutase changes the superoxide anions to hydrogen peroxide (H₂O₂) [8]. This hydrogen peroxide is further involved in the generation of other ROS. Membrane-bound nicotinamide adenine dinucleotide phosphate (NADPH) oxidase is also involved in the generation of ROS in mitochondria [9, 10].

Another significant internal source of ROS is the cytoplasm, where superoxide anions pass through voltage-dependent anion channels and are converted into hydrogen peroxide. Further, the hydrogen peroxide is converted to hypochlorous acid by myeloperoxidase present in the cytoplasm. Hypochlorous acid is highly destructive. Cytoplasmic enzymes like cyclopropane oxygenase, xanthine oxidase, amino acid oxidase and lipid oxygenase are also involved in the production of hydrogen peroxide [9]. Cytochrome P450, activated inflammatory cells, peroxisomal metabolism, monoxidases, microsomes etc. are also involved in the production of endogenous ROS.

Exogenous factors like ionization radiations (X-rays etc.), Ultraviolet radiations, cigarette smoking, and pollutants can induce the production of ROS [9]. A diagrammatic representation of the sources of ROS and its effect on the cells is shown in Fig. 1.

Fig. 1.

Diagrammatic representation of sources of reactive oxygen species and their effect on cell

Importance of reactive oxygen species (ROS) in cancer

Basically, ROS are the byproduct of the various physiological and cellular processes. Characteristically, all the cancer cells exhibit higher than normal or baseline levels of ROS. Aerobic respiration results in the creation of ROS along with Superoxide dismutase, Hydrogen peroxide and Glutathione. However, in cancer cells, the levels of these byproducts of aerobic respiration are higher than the normal cells. Levels of glutathione are almost four times higher in cancer cells in comparison to that found in normal cells [1]. High levels of glutathione are also responsible for the drug resistance against cancer therapy.

Alterations in cellular metabolic pathways, genetic alterations, and hasty proliferation are some of the major contributory factors for the elevated levels of ROS in cancer cells [11]. ROS contributes to both cancer cell proliferation and progression. Nevertheless, cancer cells acquire the capability to regulate ROS levels, maintaining them below cytotoxic levels but sufficiently high for promoting tumor growth. This redox balance is the hallmark of cancer cells. Also, this redox balance is one of the primary reasons for the induction of drug resistance in cancer cells and cancer progression as well as its relapse [11]. An increase in ROS beyond redox homeostasis can induce apoptosis and cell death even in cancer cells.

Certain mechanisms have been proposed for the high ROS levels in cancer cells [11]-

• Rapid proliferation in the cancer cells demands high energy which leads to hypermetabolism. The hypermetabolism in cancer cells causes excessive generation of ROS in the Endoplasmic reticulum, mitochondria and cellular membrane.

• Mutation in mitochondrial DNA leads to defective electron transport chain resulting in excessive production of ROS.

• Certain cancer cell-specific mutations bring about changes in the pathways of intracellular cell signalling which alters cellular protein production and metabolism. This also results in the accumulation of intracellular ROS in cancer cells.

• Hypoxia in the cancer or tumour cell microenvironment also stimulates the production of ROS.

• External factors or environmental stimuli like, cigarette smoking, exposure to ultraviolet radiation, pollutants and alcohol have been found to be carcinogenic due to their ability to induce high levels of ROS production.

Thus, cancer cells, characteristically maintain ROS levels well above basal levels of non-cancerous cells but at the same time well below cytotoxic levels. This characteristic redox balance helps the cancer cells in the initiation of tumorigenesis, proliferation or growth of cancer cells, propagation and eventually metastasis of cancer. High ROS in the tumour microenvironment causes immune suppression and oxidative stress that results in the premalignant transformation of cells that initiate the tumour formation [12]. Non-cancerous stromal fibroblast and epithelial cells undergo a premalignant transformation in the presence of chronic elevated levels of hydrogen peroxide (H₂O₂) [13]. Upregulation of MAPK/ERK and PI3K/Akt/mTOR signalling cascades occurs due to ROS-induced oxidised and inactivated phosphatase which results in the initiation of tumorigenesis [14, 15]. Further, endogenous ROS induces the vascular endothelial growth factor (VEGF)–mediated angiogenesis in the tumour cells thus promoting tumour growth [16]. High ROS levels also help in cancer metastasis by modifying cell–cell junction destabilization, increasing the mobility of cells, alteration and reshaping of cellular cytoskeleton and extra-cellular matrix [11].

Cancer cells thus maintain higher than basal levels of ROS; remarkably the levels are well below the cytotoxic level with the help of antioxidant enzymes. To sustain redox homeostasis, cancer cells upregulate the production of antioxidant enzymes like glutathione, peroxiredoxin and thioredoxin. This is also referred to as the redox adaptive mechanism of cancer cells that also helps the cancer cells to develop anticancer drug resistance [7].

Classification of ROS- sensitive nanomaterials

One of the distinguishing features between cancer cells and normal cells, is the level of ROS or the Redox potential. This has fuelled the development of ROS-sensitive nanoparticles for cancer therapy. Broadly, ROS-sensitive nanoparticles can be classified into three broad categories [17]-

• Inorganic ROS-sensitive nanomaterials

• Organic ROS-sensitive nanomaterials

• Organic–inorganic hybrid ROS-sensitive nanomaterial

Inorganic ROS-sensitive nanomaterials

As the name suggests, such ROS-sensitive nanomaterials are inorganic carriers like metals and their oxides, carbon nanomaterial and mesoporous silica. Inorganic nanomaterials offer advantages like stability, cost-effectiveness, easy scalability, easy surface modulation for achieving site-specificity, and easy tunability in terms of size and shape [18, 19]. The drug release from inorganic nanomaterials can be modulated and controlled by inducting oxidation- or reduction-receptive bonds [18]. Some of the redox-sensitive bonds include the diselenide bond (i.e., -Se-Se-), the Disulfide bond (i.e., -S–S-), and the thioether bond. The diselenide bond is a weak bond which exhibits high sensitivity to even low amounts of oxidative agents [20]. Diselenide bond is usually used in di-block copolymers and as a crosslinking agent. Diselenide bonds are used to combine the hydrophilic unit with that of the hydrophobic unit [21]. Similarly, the disulphide bond is also a redox-sensitive chemical bond and in the vicinity of a reducing agent it gets converted to the thiol group. While thioether bonds get cleaved by the oxidizing agents to generate either hydrophilic sulphone or sulfoxide [22]. Interestingly, as compared to disulphide bonds, diselenide bonds are low-energy, weak bonds [20].

Metal and metal oxide ROS-sensitive nanomaterials

Examples of metal and metal oxide-based ROS-sensitive nanomaterials include gold-based nanoparticles, iron oxide nanoparticles, cerium-based nanoparticles, titanium-based nanoparticles, copper-based nanoparticles, as well as silver and platinum-based nanoparticles. These diverse materials are being actively researched for their unique properties in various applications, particularly in biomedical fields, where their ability to respond to reactive oxygen species (ROS) is of significant interest. Such nanomaterials offer promising potential in targeted drug delivery, imaging, and other therapeutic interventions. These metal and metal oxide nanoparticles are biocompatible, physical stability and optical characteristics [23].

Gold nanoparticles

Gold nanoparticles are ROS-catalysing nanoparticles that stimulate the production of ROS. Interestingly, Gold nanoparticles do not exhibit the property of redox sensitivity, but they are known to result in the production of ROS by increasing the transfer of electrons from the electrosensitive biological molecules. This results in an increase in lipid peroxidation and ROS levels which damages the double-stranded DNA and eventually causes cell apoptosis [5]. Due to the sensitivity of gold nanoparticles for ROS, they are also used for the detection and estimation of ROS like hydrogen peroxide (H₂O₂) and superoxide anion (O₂•-) [24, 25]. Additionally, gold nanoparticles also possess’ characteristic optical properties due to which they are widely used for photodynamic therapy (PDT). Thus, a combination of ROS triggering property and optical property makes gold nanoparticles an efficient candidate for PDT [26, 27]. Further, the surface of gold nanoparticles can also be modified to make it ROS-sensitive by the addition of redox-sensitive ligands or functional groups, which can easily be cleaved by reducing agents like glutathione (GSH) or photothermal energy. One such polyplatinum (IV)-coated gold nanorod (GNR@polyPt(IV)) that was redox responsive were developed by in-situ polymerization. These gold nanorods exhibited redox-responsive drug release in the tumour cells and amplified tumour accumulation on the application of mild hyperthermia induced by near-infrared (NIR) [28].

Iron oxide nanoparticles

Iron oxide nanoparticles are the most widely investigated metal oxide nanoparticles due to their distinct superparamagnetic property for application in targeted drug delivery [29], magnetically assisted transfection [30] and imaging through magnetic resonance imaging (MRI) [31]. However, iron oxide particles are also ROS-sensitive nanomaterials based on Fenton and Haber–Weiss reactions [32]. With these reactions, iron ions (Fe⁺² or Fe⁺³) in the iron oxide nanoparticles, generate ROS that results in oxidative stress, eventually leading to DNA damage and mitochondrial and lysosomal dysfunction. In acidic or low pH conditions, iron oxide nanoparticles exhibit peroxidase-like activity to promote the oxidation of peroxidase which results in the generation of ROS [33]. The presence of hydrogen peroxide in tumour cells further acts synergistically with iron oxide nanoparticles to generate toxic hydroxyl radicals (OH•). These hydroxyl radicals are highly useful in cancer therapy. Typical Fenton reactions are [34].

$${\text{Fe}}^{2+}+{\text{H}}_2{\text{O}}_2\underset{\text{Fe}}{⟶}+·\text{OH}+{\text{OH}}^{-}$$

$${\text{Fe}}^{3+}+{\text{H}}_2{\text{O}}_2\underset{\text{Fe}}{⟶}+·{\text{HO}}_2+{\text{H}}^{+}$$

A study reported the synthesis of amorphous iron nanoparticles (AFeNPs) that exhibited highly cancer cell-specific therapy by generating hydroxyl radicals. These amorphous iron nanoparticles were found to be more efficient than crystalline iron nanocrystals. Amorphous iron nanoparticles and the hydrogen peroxide, present in the tumour microenvironment, stimulate the production of hydroxyl radicals via the Fenton reaction. The resultant ROS puts the cell under oxidative stress leading to cellular apoptosis of the cancer cells [35]. In another study, core–shell iron oxide nanoparticles were synthesised and were found to possess higher stability and efficiency in generating ROS under acidic conditions and in the presence of tumour cell hydrogen peroxide. The overproduction of hydrogen peroxide in tumour cells creates a cycle of ROS production in an exponential pattern by the core–shell iron oxide nanoparticles. Thus, core–shell iron oxide nanoparticles were found to be a safe and efficient modality in targeting cancer cells [36].

Cerium-based nanomaterials

Nanomaterials based on Cerium, a rare earth metal, have been found to be effective in cancer therapy due to their ability to generate ROS. Cerium is a highly reactive metal with the catalytic property of its ions, Ce⁺² and Ce⁺³ [5]. The cerium oxide nanoparticles have both the capability of being an antioxidant as well as generating ROS due to their redox surface properties [37, 38]. Cerium oxide nanoparticles induce oxidative stress that leads to apoptosis specifically in cancer cells while sparing normal cells. Research has demonstrated that these nanoparticles possess the ability to target cells selectively. In an invitro study, nanoceria or cerium oxide nanoparticles were found to enhance ROS production in fibrosarcoma tumour cells in a dose dependant manner and eventually result in tumour cell apoptosis. Notably, the normal cells remain unaffected even at the high concentration of the nanoceria [39]. Further, western blot analysis and real-time PCR shoed that nanoceria increase the expression of Bax in fibrosarcoma tumour cells thus confirming the antitumor activity of nanocerium [40]. Cerium oxide nanoparticles are also known to exhibit antioxidant properties. In one of the studies, cerium oxide nanoparticles were found to consume hydrogen peroxide of the tumour cells to generate molecular oxygen in the tumour cells [41, 42]. This opens up the utility of cerium oxide nanoparticles in combination with PDT wherein along with the generation of oxygen in the tumour microenvironment, irradiation with near IR radiations leads to the creation of superoxide anions (O₂∙−) and hydroxyl radicals (•OH) by cerium oxide nanoparticles. These elevated levels of ROS result in cellular apoptosis in tumour cells [43].

Titanium-based nanomaterials

Titanium-Based nanomaterials have been found to produce excess ROS on exposure to light thus making them a suitable candidate to be used in conjunction with PDT [44]. Like, other metal oxide nanoparticles, titanium oxide nanoparticles result in oxidative stress and eventually DNA damage due to their ability to generate ROS. Studies have shown titanium oxide nanoparticles to be effective in cervical cancer, non-small-cell- lung cancer, breast cancer and colon cancer in animal models [45–47].

Copper based nanomaterials

Copper-based nanomaterials are affordable and highly versatile as compared to iron oxide nanomaterials. This is due to the efficiency of copper to catalyse the Fenton reaction to convert hydrogen peroxide to hydroxyl radicals (•OH), over a broad pH range [5]. One study reported the rate of reduction of hydrogen peroxide by copper ion (Cu⁺²) to be 4.6 × 102 M⁻¹ s⁻¹ as against the rate of reduction by iron ion, Fe³⁺ ion, to be 0.001–0.02 M⁻¹ s⁻¹ [48]. This clearly shows the rapid rate of reduction of hydrogen peroxide to generate hydroxyl radicals by copper oxide nanomaterials, thus making it a preferred candidate for oxidative cancer treatment. Study findings have shown that copper oxide nanomaterials have been found to be more efficacious in cancer therapy as compared to iron oxide nanoparticles [49].

Carbon nanomaterial

Carbon nanomaterials like, graphene, carbon nanotubes and fullerene are well-established target-specific nanocarriers due to their distinct properties like large surface area, physicochemical properties and exceptional functionalization modality. However, the antimicrobial and antibacterial properties of graphene oxide and reduced graphene oxide have been attributed to its peroxidase-like redox ability. The structure of graphene oxide has multiple oxygen defect sites wherein the molecular oxygen gets entrapped and later converted to ROS, thus causing cell death elucidated as an antimicrobial effect [50, 51]. On the other hand, fullerene has been found to exhibit antioxidant properties (like, superoxide dismutase) wherein it scavenges the ROS due to the occurrence of multiple double bonds in its structure [52]. Thus, fullerene has been shown to protect against the cellular damage caused by oxidative stress [53].

Other inorganic nanomaterials

Mesoporous silica is widely used by modifying its surface characteristics by introducing redox-sensitive cleavable bonds which impart redox characteristics to them. In a study, a redox-sensitive disulphide bond was introduced, which can be cleaved by glutathione, on the silica backbone of the mesoporous silica nanoparticle to deliver doxorubicin and siRNA to the tumour cells [54].

Organic ROS-sensitive nanomaterials

Potential cytotoxicity, owing to the issue of non-biodegradability, of the inorganic ROS-sensitive nanomaterial is one of the major concerns that has directed the development of organic ROS-sensitive nanomaterial. On the other hand, organic ROS-sensitive nanomaterials are biocompatible and biodegradable and hence are being highly explored for cancer therapy. Some of the organic nanomaterials that have been explored over the years include micelles, liposomes, dendrimers, nanogels etc. Liposomes are one of the most studied organic nanoparticle systems that are easy to develop and exhibit biological compatibility due to their phospholipidic composition. ROS-sensitive liposomes are prepared by their surface modification by the introduction of redox-sensitive bonds like disulphide bonds, thioether bonds, diselenide bonds, and thiol groups. Such ROS-sensitive liposomes release drugs due to cleavage of redox-sensitive bonds like disulphide bonds and destabilization of liposomal membranes [55–57].

A similar approach has been utilized to develop ROS-sensitive nano gels wherein, redox-sensitive bonds are introduced in the nano gel network or a combination of the drug with the metallic nanoparticle is entrapped in the nano hydrogel system. The hydrogel system then acts as a reservoir for the drug and releases the chemotherapeutic for a prolonged duration [58, 59]. In one such study, a nanocomposite of copper, silver and hemin was entrapped in the agarose hydrogel. The antioxidant (glucose oxidase) like property of silver nanomaterial results in the consumption of glucose in the tumour microenvironment resulting in the production of hydrogen peroxide. The reaction between copper, silver and hemin nanocomposite and hydrogen peroxide results in the generation of ROS that inhibits tumour growth in vivo in animals [59]. Similarly, in another study, a nano enzyme hydrogel was used as a reservoir for luminogen that exhibits controlled release of the drug for a prolonged duration. Persian blue nanoparticles along with luminogen were entrapped in the nano hydrogel of agarose. Persian blue nanoparticle consumes the hydrogen peroxide from the tumour environment to release molecular oxygen. In the presence of a sufficient amount of molecular oxygen, luminogen then generates high levels of ROS leading to tumour cell cytotoxicity [60].

ROS-sensitive polymeric micelles are prepared using disulphide bond modification to achieve site-specific targeting [61]. A similar approach is utilized to develop ROS-sensitive dendrimers [62].

Organic–inorganic hybrid ROS-sensitive nanomaterial

A hybrid or composite nanomaterial comprised of both organic and inorganic components forms ROS-sensitive nanoparticles that work synergistically. This combination enhances tunability, improves site-specific targeting, optimizes drug pharmacokinetics, increases bioavailability, and ultimately leads to better therapeutic outcomes. The metal–organic framework is the most common hybrid nanomaterial design wherein the metal ion is combined with the organic ligand. A coordinate composite of ferric and ferrous irons with cyanides is utilized in the formation of Prussian blue nanomaterial [63]. Prussian blue nanomaterial possesses antioxidant activities similar to catalase, and superoxide dismutase that scavenge the ROS. Additionally, Prussian blue nanomaterial also has activities similar to peroxidase that results in the generation of ROS [64].

On a similar framework, Mn(III)-sealed metal–organic nanomaterial was prepared using porphyrin (TCPP) and Mn(III). Glutathione present in the tumour microenvironment cleaved the nanomaterial to release Mn(III) and TCPP. This also results in the consumption of glutathione from the tumour microenvironment. The resultant free Mn(III) and TCPP which help in magnetic resonance imaging and fluorescent imaging of the tumour cell [65].

Approaches of ROS-sensitive therapy

Characteristically, cancer cells maintain a critical redox balance between antioxidant and pro-oxidant levels so that cancer cells do not undergo apoptosis even when under higher ROS levels. Thus, any imbalance induced in this redox balance by any exogenous agent can result in exceeding the cytotoxic threshold and induction of apoptosis in cancer cells. A diagrammatic illustration of the mechanistic approaches of ROS-sensitive nanomaterial is shown in Fig. 2.

Fig. 2.

Diagrammatic representation of mechanistic approach of ROS-sensitive nanomaterial in cancer therapy

To eliminate cancer cells, there are two possible contradictory approaches available [66]-

• Usage of Antioxidant nanostructures wherein the nanomaterial acts as an antioxidant enzyme and scavenges the ROS [Table 1].

• Usage of Pro-oxidant nanostructures wherein, the nanomaterial acts to increase the levels of ROS in the cancer cells well above the cytotoxic levels to induce apoptosis in the cancer cell. The excessive ROS levels result in cellular oxidative stress that leads to protein and DNA damage, eventually resulting in cancer cell apoptosis [Table 1].

Table 1.

The mechanistic approach of different ROS-sensitive nanomaterials in cancer therapy

S. No	ROS-sensitive Nanoparticle	Mechanism of action	Therapeutic approach	References
1	Copper based nanoparticle	Simulate enzyme-like activity ~ catalase, peroxidase, glutathione and superoxide dismutase	Antioxidant	[67, 68]
2	Gold-based nanoparticles	Exhibit glucose oxidase-like activity	Proxidant	[59]
3	Platinum-based nanoparticles	Simulate enzyme-like activity ~ catalase, peroxidase, superoxide dismutase	Antioxidant	[69, 70]
4	Cerium oxide nanoparticles	Exhibit catalase and superoxide dismutase-like activity	Antioxidant	[71, 72]
5	Prussian blue-based nanoparticles	Exhibit catalase, peroxidase, superoxide dismutase-like activity	Antioxidant as well as prooxidant	[73, 74]
6	Fullerene	Scavenge ROS due to the occurrence of multiple double bonds in its structure	Scavenger of ROS	[75–77]

Application of ROS- sensitive nanomaterial for cancer therapy

ROS- sensitive nanomaterials as drug delivery vehicle

Usage of the ROS-sensitive functional is commonly employed to develop ROS-sensitive nanomaterial that offers site-specific targeting. Oxidation of sulphide from + 2 to + 6 state occurs in the oxidizing environment resulting in the conversion of hydrophobic sulphide to the hydrophilic sulfones or sulphoxides. This approach has been utilised for developing ROS-sensitive micelles, of an amphiphilic polymer, poly(propylene sulfide)-polyethylene glycol-serine-folic acid (PPS-mPEG-Ser-FA). Anticancer drug, doxorubicin along with zinc phthalocyanine (a photosensitizer) was encapsulated within PPS-mPEG-Ser-FA micelles having a diameter ~ 80 nm. On exposure to laser light and the physiological hydrogen peroxide of the tumour microenvironment, the poly(propylene sulfide) segment of PPS-mPEG-Ser-FA is converted to hydrophilic sulfoxide, thus dismantling the micelles to release doxorubicin in the tumour cells. Laser light irradiation of zinc phthalocyanine stimulates the production of ROS resulting in concurrent antitumour effect of doxorubicin along with oxidative stress [78]. With a similar approach, in another study, ROS-sensitive nanoparticles of paclitaxel and chlorin e6 (Ce6) were prepared using chondroitin sulfate-g-poly (propylene sulfide). A polypropylene sulphide chain is added to the chondroitin which gets oxidised when ROS are produced by the Ce6 upon irradiation with light. This leads to the disintegration of the nanoparticle to release the anticancer drug paclitaxel in the tumour cell [79].

ROS-sensitive polymeric nanocapsules were prepared by using thioether linkage between oligo(ethylene glycol) (OEG) and 7-ethyl-10-hydroxyl-camptothecin (SN38). Thioether linkages get oxidised under high ROS levels leading to the formation of hydrophilic sulfones or sulphoxides. This leads to breakage of nanoparticles to release the drug in the tumour cells. In vitro studies show that on exposure to hydrogen peroxide or light, these ROS-sensitive nanocapsules exhibit rapid release of the drug, thus confirming the ROS sensitivity of these nanocapsules [80].

Tellurium-containing polymers are used for ROS-sensitive nanoparticle formation due to the property of tellurium to readily undergo oxidation from a lower valency (+ 2) state and hydrophobic state to a higher valance state (+ 4 or + 6) and hydrophilic state. This is similar to sulphides and selenium-containing polymers. Based on this, in a study, photosensitive porphyrin was encapsulated in a polyelectrolyte multilayer through layer-by-layer self-assembly using poly(styrene sulfonate) and tellurium-containing polymer. Upon irradiation, photosensitive porphyrin generates ROS which oxidises tellurium to a tellurium carbonyl group. This results in the disintegration of nanomaterial and the release of the anticancer drug [81].

In another study, cisplatin and indocyanine green were encapsulated in a nanocarrier made up of block copolymer of tellurium with polyethene glycol (PEG) i.e., PEG-PUTe-PEG. Indocyanine green is a photosensitive agent which upon irradiation with near IR radiation results in the formation of ROS. The generation of ROS causes the oxidation of tellurium and the formation of the tellurium carbonyl group which destabilises the block copolymer, PEG-PUTe-PEG, to release the drug cisplatin. Thus, a synergistic effect of cisplatin with indocyanine green provides an efficient way to target tumour cells [82].

Similar to tellurium and sulphide, selenium-containing nanomaterial also oxides hydrophobic selenium to hydrophilic selenides. Moreover, diselenide bonds are cleaved both under oxidising as well as reducing environment. This property was studied in a study wherein a block copolymer of selenium with PEG was studied in the presence of water and hydrogen peroxide. In the copolymer, PEG-PUSeSe-PEG, a diselenide unit is linked to PEG units. In the presence of water, PEG-PUSeSe-PEG undergoes self-assembly to form nano micelles, however, incubating the micelles with hydrogen peroxide results in the disintegration of micelles. This confirms the sensitivity of selenium-containing to hydrogen peroxide or ROS [83]. ROS-sensitive nanomaterial was prepared using selenium-containing polymer which undergoes depolymerization in a controlled manner via ROS-mediated selenoxide elimination reactions. Selenium sulfoxide is generated from the polymeric main chain selenium in the presence of ROS [84]. Doxorubicin-loaded ROS-sensitive nanoparticles (I/D-Se-NPs) were prepared using selenium copolymer, which underwent rapid oxidation to generate ROS when exposed to near IR radiations. This results in nanoparticle disintegration and eventually release of doxorubicin [85].

The ROS has also been utilized for the cleavage of thioketal linkage in the polymer which results in sulfhydryl groups and acetone. Such polymers are being utilised for the development of ROS-sensitive nanoparticles. A cationic linear polymer poly(amino thioketal) (PATK) polymer was used to form a plasmid DNA complex (< 200 nm in size) for the delivery of DNA plasmid in the tumour cell. Once internalised, the thioketal bond, in the DNA complex of PATK, is cleaved by the hydrogen peroxide present in the tumour cells and releases the plasmid DNA [86]. A poly prodrug of mitoxantrone (MTO) was developed with a ROS-sensitive thioketal linker. The poly-prodrug nanoparticles were prepared using lipid-polyethylene glycol (lipid-PEG). Further, for improved permeation through tumour endothelium one internalizing peptide (iRGD), is added to the poly prodrug nanoparticle. Under ROS, the thioketal linker undergoes cleavage to release the drug in the tumour cell [87]. Thioketal linkage was utilised to form a multifunctional prodrug of Gemcitabine in combination with meso-tetraphenyl porphyrin (TPP), a fluorescent photosensitizer. Upon irradiation, TPP generates ROS, singlet oxygen, to cause damage to the tumour cells. Concurrently, in the presence of ROS, thioketal linkage gets cleaved to release the Gemcitabine which also causes tumour cell cytotoxicity [88].

In a similar pattern, hydrogen peroxide selectively breaks the aryl borate bonds which results in the formation of boric acid and phenol. Based on this, phenylboronic acid pinacol ester-functionalized PEGylated copolymer methoxyl polymer mPEG-b-P(PA-alt-GPBAe) was utilised to encapsulate doxorubicin. At low pH and in the presence of hydrogen peroxide, the phenyl borate ester of the copolymer undergoes oxidation and disassembly of the nanoparticle to release the drug in the tumour cell [89]. A ROS-sensitive core–shell type polymeric microgel of the anticancer drug, 5′-deoxy-5-fluorocytidine (DFCR) was prepared wherein the core–shell was made by linking poly(hydroxyethyl methacrylate) (poly-HEMA) with the pheophorbide A (PheoA), a photosensitizer. This core–shell was further encapsulated with in a chitosan layer to make CSPM-PheoA-DFCR hydrogel. Upon irradiation, this CSPM-PheoA-DFCR hydrogel stimulated the release of ROS that caused cytotoxicity as well as the cleavage of the phenylboronic ester linker between the DFCR and the chitosan shell. The cleavage resulted in the release of the drug to result in tumour cell cytotoxicity [90].

Diethylstilbestrol was linked with polyoxalate skeleton, which in the presence of ROS undergoes cleavage to release the drug [91]. A ROS-sensitive hydrogel fo doxorubicin was developed with tetra-poly(ethylene glycol)-b-oligo (l-methionine) (t-PEG₅₆-b-OMeth_n) which contains poly(L-methionine). Under high ROS levels, the side chain of L-methionine (hydrophobic) undergoes oxidation to form hydrophilic methionine sulfoxide. This leads to the release of the drug, doxorubicin in the tumour cells [92]. In one of the studies, a therapeutic protein caspase-3 was encapsulated in a polymeric nanocapsule connected via a disulphide bond with a crosslinker, N, N′-bis(acryloyl)cystamine. Under the reducing conditions, cleavage of the disulphide bond occurs leading to the release of the caspase-3, which induced apoptosis in cancer cells like, MF-7, HeLa and U-87 [93]. In another study, for the cytosolic delivery of doxorubicin, a folic acid-linked polymer, having a disulphide bond, was coated upon lipid nanoparticles having acoustic reflective properties. In oxidising conditions, the nanoparticles were stable however, upon exposure to reducing conditions, nanoparticles disintegrate and release the drug. The expression of folate receptors is amplified in the tumour cells hence, the presence of folic acid present on the nanoparticle also helps in achieving cancer-site specificity. Also, application of diagnostic frequency ultrasound further enhances the drug release [94].

Use of ROS-sensitive nanomaterial in combination with other anticancer therapies

Usage of photodynamic therapy (PDT), chemodynamic therapy (CDT) and sonodynamic therapy (SDT) are also used for cancer therapy as exposure to these therapies also results in the elevation of ROS in the tumour cells. Thus, to increase the efficiency and site specificity along with the synergistic effect, ROS-sensitive nanomaterials are being used in combination with PDT, CDT, and SDT [95].

ROS-sensitive nanomaterial in combination with chemodynamic therapy

Chemodynamic therapy utilizes the Fenton/Fenton-like reaction that occurs between metallic ions and hydrogen peroxide, in the absence of any external energy source like radiations, to generate hydroxyl radical (^•OH) or ROS [34]. In other words, the generation of oxidative stress and toxic ROS due to the Fenton/Fenton-like interaction between hydrogen peroxide and metal ions. These toxic ROS hydroxyl radicals eventually cause apoptosis or cytotoxicity in tumour cells. CDT has the advantage of lower systemic side effects, low cost and convenience, however, dependency on endogenous hydrogen peroxide, to induce Fenton/Fenton-like reaction and eventual generation of hydroxyl radical is, makes CDT unpredictable. Hence, to improve the efficiency and reproducibility of CDT, a combination of ROS-sensitive nanomaterial and CDT is being employed [96]. The approaches to improve the efficiency of CDT with ROS-sensitive nanomaterial include [96]-

• Increasing the amount of hydrogen peroxide at the tumour site

• Scavenging glutathione at the tumour site

• Intensify the hydroxyl radicals in the tumour site

• Usage of external energy

To improve upon the therapeutic efficiency of the dendrimer-encapsulated doxorubicin, a metal–phenolic network of Fe⁺³ and tannic acid is introduced on the dendrimer. The presence of Fe⁺³ ion increases the ROS by Fenton/Fenton-like reaction, thus enhancing the efficacy of doxorubicin released from the dendrimer [97]. In another study, a combination of glucose oxidase and manganese-doped calcium phosphate is loaded upon the doxorubicin nanoparticle. Glucose oxidase scavenges the intratumoral glucose which leads to the generation of hydrogen peroxide. The hydrogen peroxide in contact with manganese induces a Fenton/Fenton-like reaction to produce toxic hydroxyl radicals resulting in the amplification of tumour cell cytotoxicity by doxorubicin released from nanoparticle [98]. Platinum is also known to stimulate the production of ROS via Fenton/Fenton-like reaction. Hence, cisplatin was incorporated in a nanoparticle containing iron oxide. As a result, cisplatin induces the production of superoxide anions which are eventually converted to hydrogen peroxide. The hydrogen peroxide produced further reacts with Fe⁺³/Fe⁺² ions to produce hydroxyl radicals, thus resulting in enhanced tumour cell cytotoxicity with minimum systemic side effects [99]. Camptothecin is a drug that is known to induce the production of hydrogen peroxide. In one study, camptothecin was encapsulated in a pH-sensitive iron oxide nanoparticle. Upon internalization in the tumour cell, low pH causes the disintegration of the nanoparticle and subsequent release of camptothecin. Camptothecin stimulates the production of hydrogen peroxide which further reacts with iron ions to produce the hydroxyl radicals. Thus, driving tumour cell cytotoxicity by inducing excess ROS in the tumour microenvironment [100]. In another study, Tirapazamine, a hypoxia-driven drug was loaded onto mesoporous silica nanoparticles with iron oxide nanoparticles and glucose oxidase grafted on the nanoparticle surface. Glucose oxidase results in the consumption of glucose and oxygen leading to acidic, hypoxic and hydrogen peroxide-rich tumour microenvironment. A hypoxic environment triggers Tirapazamine while iron oxide generates toxic hydroxyl radicals resulting in tumour cell cytotoxicity [101].

Thus, employing ROS-sensitive nanoparticles along with CDT results in efficient anticancer therapy with reduced systemic toxic effects.

ROS-sensitive nanomaterial in combination with photodynamic therapy (PDT) and sonodynamic therapy (SDT)

The three basic components of PDT are photosensitizer, oxygen and irradiating light source. Some of the common photosensitizers are- porphyrins, phthalocyanines and chlorins [102]. PDT involves the activation of a photosensiting agent by external irradiation. the activated photosensitizing agent acquires the energy and moves from the ground state to the first excited state (¹PS•) and eventually to the triple excited state (³PS•) through a series of transitory steps. The triple state has a long life and is capable of inducing cytotoxicity via two possible mechanisms [34]-

• Type I, wherein the transfer of electron from triple state results in the formation of radicals which further react with molecular oxygen to generate ROS.

• Type II, wherein, electron transfer results in the formation of singlet oxygen (.¹O₂)

The hypoxic tumour microenvironment has been found to reduce the efficiency of PDT, hence PDT has been combined with the ROS-sensitive nanomaterials. In line with this, protoporphyrin IX linked with zinc was loaded onto the protein bacterioferritin (ZnP-Bfr) having iron storing capacity. The inner core of the nanoparticle was filled with ferric oxyhydroxide polymer and conjugated with the outer surface through polyethene glycol. Upon irradiation, zinc protoporphyrin stimulates the production of hydroxyl and singlet oxygen radicals. While Fe⁺³ is reduced to Fe⁺² in reaction with hydrogen peroxide and generating hydroxyl radical in this process. Thus, the dual modality of the nanoparticle increases the efficiency of the therapy [103].

A hydrophobic photosensitizer 5,10,15,20-tetrakis(4-methacryloyloxyphenyl)porphyrin (TMPP) is loaded onto ferrocene-containing amphiphilic block copolymer (PEG-b-PMAEFc) nanoparticle (TPFcNP). Characteristically, TMPP contains multiple double bonds. Under acidic conditions, the ferrocene acts as a catalyst to promote the generation of hydroxyl radicals from hydrogen peroxide. The hydroxyl radical then catalyses the reaction between TMPP and endogenously overexpressed glutathione (GSH) to increase the solubility or hydrophilicity of TMPP. Increased hydrophilicility also reduces the possibility of aggregation of the photosensitizer which improves the efficiency of the photosensitizer in the therapy. Thus, concomitant usage of ROS-sensitive nanomaterial can be utilised to improve the efficiency of PDT therapy [104].

In another study, zinc phthalocyanine (ZnPc) was loaded onto nanoparticles of ferric pyrophosphate (FeP-ZnPc). The Fe⁺³ from FeP is released to react with the endogenous GSH to form Fe⁺², which further catalyses the Fenton reaction to produce hydrogen peroxide. While upon irradiation, ZnPc gets stimulated to produce the ROS, thus, the two approaches act in conjunction to improve the efficiency of the therapy [105].

The fundamental principle of SDT resembles that of PDT, with the photosensitizer being substituted by a sonosensitizer. While the precise mechanism of SDT is not fully understood, it is generally accepted that acoustic cavitation plays a role in the production of ROS during SDT [34]. Ultrasound offers better patient compliance, deep tissue penetration and control. However, like PDT, the efficiency of SDT is reduced due to hypoxia in the tumour microenvironment [102, 106]. Hypoxia has been found to reduce the amount of ROS released by SDT [107].

A multifunctional nano sonosensitizer was developed in combination with the MnO_x component and hollow mesoporous organosilica nanoparticles. This construct was then combined with the sonosensitizer (protoporphyrin) and cyclic arginine-glycine-aspartic pentapeptide. The MnO_x converts the tumorogenic hydrogen peroxide to molecular oxygen, thus acting as a nanoenzyme to improve the oxygenation state for better efficacy of SDT [108].

In a study, poly(ethylene glycol) (PEG) and stearine were linked via a thioketal bond to form a ROS-sensitive polymer which was used to encapsulate doxorubicin along with a photosensitizer pheophorbide A (PhA). Upon internalization, the initial release of doxorubicin and PhA from the nanoparticles was driven by the high ROS levels of the tumour cells. However, upon irradiation, PhA stimulates and drives the production of the singlet oxygen (¹O₂), which in conjunction with the doxorubicin hastens the cell cytotoxicity of the tumour cells [109].

ROS -sensitive nanomaterial in combination with radiation therapy (RT)

The use of high-energy radiation, such as X-rays and gamma radiation, leads to the generation of ROS, which then induces DNA damage in the targeted tumor cells. The use of ROS-sensitive nanomaterial when used in conjunction with RT results in cell-specific drug release and efficient drug therapy [34]. A ROS-sensitive nanoparticle (PEG–PSDEA–PEG) of polyethene glycol (PEG) with poly(thiodiethylene adipate) (PSDEA) encapsulating camptothecin analogue, SN38, in it. In the high ROS conditions, created by exposure to X-rays, in the tumour cells, nonpolar sulfide residue is oxidised to polar sulfoxide units, which disintegrate nanoparticles to release the drug. This delivery system exhibits high antitumour activity as compared to monotherapy in the animal model [110].

The most common problem associated with radiation therapy is its severe side effects on the normal cells. Thus, to avoid cytotoxicity of the normal cell on application of X-rays, a smart nanoparticle having dual sensitivity to ROS as well as X-rays was developed using Cu₂(OH)PO₄. Exposure to the X-rays causes the generation of Cu^I due to photoelectron transfer. Later, activated Cu^I catalyse the conversion of tumorigenic hydrogen peroxide to hydroxyl radicals, leading to tumour cell cytotoxicity. Normal cells do not undergo a Fenton-like reaction due to their higher oxygenated state and low levels of hydrogen peroxide in them. Thus, utilising this approach of ROS-sensitive nanomaterial application with RT can help to minimise the toxic side effects in the neighbouring normal cells, upon application of X-rays during cancer therapy [111].

Challenges and future prospects of ROS-sensitive nanomaterials

One of the major challenges in the development of ROS-sensitive nanomaterials is the availability of sensitive, accurate and reproducible detection methods for the estimation of ROS levels [112, 113]. Currently, direct or indirect approaches (like spectrophotometry, fluorometry etc.) are used to estimate ROS levels, however, shorter life spans of ROS and their extremely low levels limit the accuracy of the method [114]. Thus, there is a need for the development of a method that can detect and estimate ROS in biological systems with accuracy, sensitivity and reproducibility.

It is a well-established fact that ROS in small quantities are essential for the basic life processes. The proxidant approach of the ROS-sensitive nanomaterials creates an imbalance in the ROS homeostasis of the tumour cell resulting in oxidative stress-induced apoptosis or cell death [115]. However, the current ROS-sensitive nano-delivery system does not act on specific ROS pathways. Stem cells of the body require certain levels of ROS for quiescence and self-perpetuation ability [116]. Thus, to maintain the ability of the body to regenerate and proliferate and reduce the side effects of cancer therapy, it becomes pertinent to develop ROS-sensitive systems that are specific to certain ROS-pathways.

Also, for the successful transition of ROS-sensitive nanomaterial to clinical therapeutic modality, ROS-nanomaterial needs to have biocompatibility and low allergenic induction potential. The ROS-sensitive nanomaterials should have better control over drug release patterns in vivo. Also, the sensitivity of the ROS-sensitive nanomaterials should be such that they remain unaffected by the ROS levels of the normal cells to avoid dose dumping and their side effects [95].

Further, ROS-sensitive nanoamaterial being ultrasmall structures can alter the ability of mitochondria to produce ROS. Thus, it needs to be ensured during their development that mitochondrial ROS production from normal cells remains unaffected. Also, ROS-sensitive nanomaterials are bound to face biological hurdles like, macrophage system, vascular flow and rheological restrictions, lysosomal and endoplasmic reticulum effects etc. Thus, studies directed towards the detailed investigation of the in-vivo effect and pharmacokinetics of ROS-sensitive nanoparticles are needed [117].

There are possibility of ROS-induced side effects and toxicity. It is well known that many currently used anticancer drug exert their cytotoxic effect by increasing the ROS levels. For instance, cardia dysfunction caused by the usage of doxorubicin is mediated by the ROS generated [118]. Similarly, carbon nanotubes, which are being explored as ROS-sensitive drug delivery vehicles have been reported to cause serious side effects invitro as well in vivo [119–122]. Platelet aggregation and granuloma formation in the lungs have been reported on the usage of carbon nanotubes [123, 124]. Similarly, dendrimers have been reported to induce immunogenic effects [125]. Some studies suggest the development of dendrimers of size < 5 nm to avoid such immune reactions [126]. The majority of the studies have concentrated their focus on the accumulation of the ROS-sensitive nanomaterial at the site of the tumour, however, the long-term effect of retention of these nanomaterial in the body needs to be studied in detail [5]. Thus, ROS-sensitive nanomaterial should be thoroughly investigated clinically for their possible side effects [7].

Conclusions

Cancer cells characteristically maintain higher levels of ROS and creating an imbalance in this homeostasis to induce tumour cell cytotoxicity is a potential approach in cancer therapy. A ROS-sensitive nanomaterial is a potential tool for achieving an efficacious and site-specific cancer therapy. An imbalance in the ROS can be induced either by scavenging the ROS from the tumour cells i.e., a prooxidant approach or by simulating the antioxidants i.e., an antioxidant approach. ROS-sensitive nanomaterials that are loaded with anticancer drugs disrupt the ROS-homeostasis in the cancer cells by using either the prooxidant or antioxidant approach resulting in oxidative stress-induced apoptosis. ROS-sensitive along with anticancer drug intensify the oxidative stress in the cancer cells thus resulting in an efficacious cancer therapy with reduced systemic toxic side effects. The use of ROS-sensitive nanomaterial along with other therapies like PDT, CDT, SDT and RT further helps to reduce the toxic effects of the cancer therapy in normal cells, thus offering a possibility of improving the quality of life of a patient. However, the successful transition of ROS-sensitive nanomaterial for clinical usage requires in-depth studies in humans regarding their pharmacokinetics on long-term usage and possible side effects associated with it. Long terms and multicentre clinical trials on humans will provide a clear picture for the effective usage of ROS-sensitive nanomaterial in clinical practice.

Author contributions

Zi Lv: Conceptualization; Weiming Huang: Writing-Original draft preparation; Eryong Zhao and Pengju Zhang: Visualization, Writing-Original draft preparation; Jian Xiong and Shaokun Wang: Validation, Software, Writing-Reviewing and Editing.

Funding

This study was funded by the Science and Technology Program of Guangzhou, China (2024A04J4520).

Data availability

No datasets were generated or analysed during the current study.

Declarations

Competing interests

The authors declare no competing interests.