Electron spin resonance spectroscopy for the study of nanomaterial-mediated generation of reactive oxygen species.

Many of the biological applications and effects of nanomaterials are attributed to their ability to facilitate the generation of reactive oxygen species (ROS). Electron spin resonance (ESR) spectroscopy is a direct and reliable method to identify and quantify free radicals in both chemical and biological environments. In this review, we discuss the use of ESR spectroscopy to study ROS generation mediated by nanomaterials, which have various applications in biological, chemical, and materials science. In addition to introducing the theory of ESR, we present some modifications of the method such as spin trapping and spin labeling, which ultimately aid in the detection of short-lived free radicals. The capability of metal nanoparticles in mediating ROS generation and the related mechanisms are also presented.

1. Introduction

Rapid development of the nanoscience and technology has produced numerous nanomaterials that offer revolutionary benefits in electronics, energy, medical, and health applications, but unfortunately also lead to environmental, health, and safety concerns [1]. For example, Au nanoparticles (NPs) have been explored as nanopharmaceuticals for the treatment of cancer [2], and Ag NPs have been established as superior antibacterial materials [3]. However, the wide use of nanomaterials has raised concerns regarding their potentially hazardous effects on biological systems, and the associated short- and long-term risks are not well understood. A variety of nanomaterials can generate reactive oxygen species (ROS) under certain experimental conditions [4-9]. Among various toxic responses, nanomaterial-induced oxidative stress mediated by ROS has been studied most extensively [10-12].

ROS, e.g., superoxide, hydroxyl radical, singlet oxygen, and hydrogen peroxide, are powerful oxidants that can damage cellular targets nonselectively. Free radicals, including ROS, are short lived and represent a broad range of chemically distinct entities; consequently, these species are difficult to detect in dynamic environments such as biological systems. The use of fluorescent probes (e.g., dichlorodihydro-fluorescein, hydroethidine, and dihydrorhodamine) and chemiluminescent assays is a simple and easy way of detecting free radicals and ROS in cellular systems, but there are inherent limitations and many sources of artifacts [13,14]. Electron spin resonance (ESR) spectroscopy has become a powerful and direct method to detect free radicals generated chemically or formed in biological systems. We have a longstanding interest in employing ESR techniques to identify and quantify free radicals in biological systems, and study the mechanisms of interactions between biologically relevant systems and nanomaterials, metal ions, and organic molecules [4,5,7,9,15-43]. We have also published several book chapters on this subject [44-46]. In this special issue, we demonstrate that ESR spectroscopy is a powerful tool for exploring the capability of NPs to generate ROS. The ESR spin-trapping techniques used to detect ROS (including hydroxyl radicals, superoxide radical anion, and singlet oxygen) and the ESR oximetry methodology employed for monitoring oxygen and the formation of lipid peroxidation are also discussed briefly.

2. ESR spectroscopy

2.1. Principle of ESR spectroscopy

ESR, also called electron paramagnetic resonance, is a powerful technique for studying chemical species or materials that have one or more unpaired electrons. The basic physical concepts of ESR are analogous to those of nuclear magnetic resonance, except that in ESR electron spins are excited instead of atomic nuclei. ESR has been studied for several decades since it was first observed by Y. Zavoisky in 1944 [47]. A number of review articles and books are available that provide a useful introduction to the basic concepts of ESR and its applications [47-49]. An electron has a spin quantum number s = 1/2 with magnetic components m = +1/2 and −1/2. In an external magnetic field, free electrons align with their spin parallel (low energy) or perpendicular (high energy) to the magnetic field (Fig. 1). A transition between low- and high-energy states can occur when sufficient energy is absorbed. This energy lies within the microwave frequencies of the electromagnetic spectrum. The energy (hv) required for this transition is given by the following equation:

Fig. 1

Energy diagram showing the origin of an electron spin resonance signal.

where μB is the Bohr magneton, B0 is the magnetic field strength, and g is the Landé g-factor (2.0023 for free electron). An ESR spectrum is usually obtained by varying the magnetic field strength at a fixed microwave frequency. Magnetic field strengths at which the microwave frequency is absorbed are recorded in the ESR spectrum. A typical continuous wave X-band (9.5 GHz) ESR instrument, as shown in Fig. 2, includes the following major components: (1) a magnet that generates and modulates a magnetic field; (2) a microwave supply system that includes an electromagnetic radiation source and a detector to control the microwave power; (3) a sample cavity to which microwave energies are directed and in which samples are placed; and (4) a data processing and display system. Under certain conditions, each free radical exhibits a specific ESR spectrum, and the intensity of an ESR signal is proportional to the concentration of free radicals; therefore, qualitative identification of free radical species along with their quantitative measurements can be performed.

Fig. 2

Photograph of a typical Bruker EMX continuous-wave electron spin resonance instrument. The components A, B, C, D, and E represent magnet, microwave supply and control system, sample cavity, data processing, and display system, respectively.

Spin trapping and spin labeling are the two principal ESR techniques used for the detection and identification of free radicals [9,44,50]. ROS are usually very reactive and present in low concentrations, which is a major limitation to the detection of ROS. However, the instability of free radicals is largely solved by the use of either spin trapping or spin labeling. The ESR spin-trapping technique uses chemical species called spin traps, which react with short-lived free radicals to form relatively stable adducts having a half-life long enough for ESR measurement [50]. The ESR spin-labeling technique uses a stable paramagnetic spin label agent to interact with the target chemical, e.g., the oxygen molecule or electrons, and is a powerful tool for probing structural and/or dynamic changes in complex chemical or biological systems [51,52]. Here, our discussion focuses on oxygen-centered free radicals, particularly ROS. In this review, carbon- and sulfur-centered free radicals have not been included.

2.2. ESR spin-trapping technique

There are three kinds of spin traps, compounds with nitrone, nitroso, and piperidine/pyrrolidine groups [50], as displayed in Fig. 3. Most of these spin traps are soluble in water and polar organic solvents, can capture a variety of free radicals, and can reveal specific hyperfine splitting information, which aids in the identification of radicals. Nitrone spin traps include cyclic and open-chain nitrones. Spin traps including 5,5,-dimethylpyrro-line N-oxide (DMPO), 5-tertbutoxycarbonyl-5-methyl-1-pyrroline N-oxide (BMPO), 5-diethoxyphosphoryl-5-methyl-1-pyrroline-N-oxide (DEPMPO), and other derivatives have similar cyclic nitrone structures and are highly useful for trapping oxygen-centered free radicals, including superoxide and hydroxyl radicals. Phenyl-tert-butylnitrone and α-(4-pyridyl N-oxide)-N-tert-butylnitrone (POBN) are common open-chain nitrone spin traps, which are typically used to trap carbon-centered free radicals. Nitrone spin traps can capture the radical at a carbon adjacent to the nitrogen, which results in the loss of chemical information. However, the most popular spin traps have a β-hydrogen that can provide considerable qualitative information about the trapped radicals. DMPO, BMPO, and DEPMPO can be used to trap hydroxyl radicals and superoxide, forming adducts with •OH or •OOH. However, in the detection of superoxide using DMPO as a spin trap, the resulting DMPO/•OOH adducts are unstable and decay to DMPO/•OH adducts, leading to a misinterpretation of the generation of hydroxyl and superoxide radicals [53]. The spin adducts BMPO/•OOH and DEPMPO/•OOH are highly stable, especially BMPO/•OOH that does not decompose into the corresponding hydroxyl adduct.

Fig. 3

Chemical structures of common spin traps and spin labels including (A) a piperidine nitroxide derivative, (B) an unsaturated pyrrolidine nitroxide derivative, and (C) a saturated pyrrolidine nitroxide derivative. BMPO = 5-tertbutoxycarbonyl-5-methyl-1-pyrroline N-oxide; CPH = 1-hydroxy-3-carboxy-pyrrolidine; DEPMPO = 5-diethoxyphosphoryl-5-methyl-1-pyrroline-N-oxide; DMPO = 5,5-dimethylpyrroline N-oxide; MNP = 2-methyl-2-nitrosopropane; NOB = nitrosobenzene; PBN = phenyl-tert-butylnitrone; POBN = α-(4-pyridyl N-oxide)-N-tert-butylnitrone; TEMP = 2,2,6,6-tetramethylpiperidine; TTBNB = 2,4,6-tri-tert-butylnitrosobenzene.

Nitroso spin traps such as 2-methyl-2-nitrosopropane and nitrosobenzene, and their derivatives, can provide more information than nitrones because the radical to be trapped adds directly to the nitroso nitrogen. However, these spin traps are not suitable for studying oxygen-centered radicals because their resulting spin adducts are photochemically and thermally unstable [53].

Representative piperidine-based spin traps, 2,2,6,6-tetramethylpiperidine (TEMP) and 4-oxo-2,2,6,6-tetramethyl piperidine (4-oxo-TEMP), can be specific traps for reacting with singlet oxygen to yield a nitroxide radical TEMPONE with a stable ESR signal, as shown in reaction (1) [9]. Consequently, TEMP has widely been used in ROS characterization for the detection of singlet oxygen. The spin trap 1-hydroxy-3-carboxy-pyrrolidine (CPH) was found to be more suitable than 1-hydroxy-2,2,6,6-tetramethyl-4-oxo-piperidine (TEMPONE-H) for trapping super-oxide radicals and peroxynitrite in biological systems [54]. CPH reacts with superoxide radicals or peroxynitrite forming the stable nitroxide radical 3-carboxy-2,2,5,5-tetramethylpyrro lidine 1-oxyl, which can be detected by ESR spectroscopy.

2.3. ESR spin-labeling technique

Spin labels are stable nitroxide free radicals, which possess an unpaired electron that has the ability to bind to another molecule. The magnetic resonance signal of this unpaired electron can be detected by ESR, which would provide information about the motion, distance, and orientation of unpaired electrons in the sample with respect to each other and to the external magnetic field. ESR spin labeling is particularly useful in the field of biology for probing local dynamics of proteins or biological membranes [55]. Most spin labels are derived from five- or six-membered hybrid rings (Fig. 3), with various functional groups indicated by R [45,56]. For example, commonly used spin labels 2,2,6,6-tetramethyl-1-piperidinyloxy, 2,2,6,6-tetramethylpiperidine 1-oxyl (TEMPO, R = H), 2,2,6,6,-tetramethyl-4-piperidone-1-oxyl (TEMPON, R = oxo), 4-hydroxy-2,2,6,6-tetramethylpiperidine 1-oxyl (TEMPOL, R = OH), and 4-amino-2,2,6,6-tetramethyl piperidine-1-oxyl (4-amino-TEMPO, R = NH2) are derived from the piperidine structure (Fig. 3A). The spin labels 3-carbamoyl-2,2,5,5-tetra-methyl-3-pyrroline-1-yloxyl (CTPO) and (1-oxyl-2,2,5,5-tetramethyl-Δ3-pyrroline-3-methyl) meth-anethiosulfonate have the same pyrroline structure with different R groups. 3-Carboxy-2,2,5,5-tetramethylpyrrolidine 1-oxyl is one representative free radical from saturated pyrrolidine structure (Fig. 3C). These nitroxide free radicals have the advantage of high sensitivity and unambiguous spectral information. A concentration of 1 μM TEMPO in a 50 μL volume can be detected easily by conventional ESR.

Spin label oximetry is a highly useful method to detect dissolved oxygen in biological environments [56]. Spin label oximetry is based on the bimolecular collision between oxygen (O2) and spin labels. As O2 is paramagnetic, a physical collision between the spin label and O2 produces a Heisenberg spin exchange, which results in a shorter relaxation time leading to a broader line width and lower peak intensity in the ESR spectrum of the spin label. Because the extent of spin exchange is dependent on the concentration of molecular O2, a change in O2 concentration results in a corresponding change in the line width of the spin label. Therefore, a real-time study of O2 generation or consumption is feasible in biological systems. The most commonly used spin labels in oximetry are CTPO and 4-oxo-2,2,6,6-tetramethyl piperidined16-1-15N-oxyl (15N-PDT; Fig. 4). Their ESR spectral shapes are dependent on the amount of O2 molecule interacting with spin labels. Practically, CTPO, because of its superhyperfine structure in the ESR spectrum, is more sensitive to O2 than 15N-PDT. The choice of spin labels is dependent on the O2 concentration. The spin label 15N-PDT is suitable for use at a high O2 concentration (>150 mM), whereas CTPO is preferred at a low O2 concentration. A limit of detection for molecular oxygen of 0.1 μM can be achieved using the CTPO spin label oximetric method [57]. By repeatedly measuring the line widths of the spin probe, one can assess the rate of lipid peroxidation in the biological sample (Fig. 4).

Fig. 4

Measurement of oxygen by ESR oximetry. (A) Oxygen consumption is measured in a closed chamber using liposome suspensions and the spin label 15N-PDT mixed with a free radical initiator of lipid peroxidation such as AAPH. (B) The black line indicates ESR spectra of CTPO in a nitrogen-saturated aqueous solution and the blue line indicates that in an air-saturated aqueous solution; the K parameter is used to determine oxygen concentration and is calculated by the equation K =(b + c)/2a. (C) The ESR spectra of 15N-PDT in a nitrogen atmosphere is shown by the red line and that in an air-saturated aqueous solution is shown by the black line. The presence of oxygen results in a broader and less intense ESR signal for the spin probe [57]. CTPO = 3-carbamoyl-2,2,5,5-tetra-methyl-3-pyrroline-1-yloxyl; ESR = electron spin resonance; 15N-PDT = 4-oxo-2,2,6,6-tetramethyl piperidine-d16-1-15N-oxyl; AAPH = 2,2′-azobis(2-amidino-propane)dihydrochloride.

3. ROS generation mediated by metal NPs

Owing to the quantum size effect, nanomaterials possess unique physiological and chemical properties that are different from those in either macroscopic (bulk) or atomic form. Concerns have been raised that these unique properties may lead to nanomaterial-induced toxicity. A variety of nanomaterials, including metal NPs, carbon nanostructures, and semiconductor NPs, have been shown to be toxic to living systems. One important mechanism of nanomaterial-induced toxicity is the generation of ROS. In this special issue, a review article by Fu et al [58], titled “Mechanism of Nanotoxicity—Generation of Reactive Oxygen Species,” discusses the mechanisms and decisive determinants of the generation of ROS by nanomaterials. As an illustration for this review, we focus our presentation on the generation of ROS mediated by metal NPs.

A variety of metal NPs have been reported to exhibit intrinsic activity in generating or scavenging ROS. Some representative results are summarized in Table 1, including NPs of metals Ag, Au, Pt, Cu, Fe, Co, Ni Fe, and Co [4-6,60-84]. Ag, Au, Cu, Fe, Ni, and Co NPs have been reported for their ability to induce the generation of ROS under certain experimental conditions. Au NPs generate ROS, including •OH, , and , in various environments [2,5,6,8,69,70], whereas Ag NPs enable the production of •OH and [4,6,8]. Cu NPs have been reported to generate •OH in the presence of hydrogen peroxide [75] and proposed to form when present with DNA in phosphate-buffered saline [76]. Most of these studies used fluorescent probes, instead of ESR, to measure ROS and reported only the total ROS production-related oxidative stress [61,62,64,67-69,73,74,82-84]. These studies indicate that physiochemical factors, including the size, shape, composition, and surface coating, of metal NPs affect ROS levels significantly. ROS production occurs mainly through two mechanisms: (1) Fenton-like reaction and (2) surface plasmon resonance enhancement; both of these are discussed in detail in the following subsections.

Table 1

Generation of ROS mediated by metal NPs, based on the data from previous reports.

NPs	ROS production	Experimental conditions and detection methods	Refs
Ag	•OH	0.5 mM H2O2, pH < 4.5, 10–100 nm, different coatings, ESR	[4]
	ROS, •OH	9–21 nm, with light, fluorescence	[60]
	ROS	In cell, 15–55 nm, fluorescence	[61]
	ROS	In human liver cell, 5–10 nm, fluorescence	[62]
	Free radicals	10 nm, ESR	[63]
	ROS	In cell, 6–20 nm, fluorescence	[64]
	O2-_•	Protein/membrane, SOD	[65]
	O2-_•	1.0 M KOH, H2O2, Al supported, ESR	[66]
	ROS	In cell, 25–70 nm, fluorescence	[67]
	ROS	Intracellular, PVP coated, 70 nm, fluorescence	[68]
	•OH, O2-_•	Under UV, fluorescence	[6]
	O21	Ag NPs, photoirradiation, fluorescence	[8]
Au	•OH	0.5mM H2O2, pH < 3.6, 10–100 nm, different coatings, ESR	[5]
	O21	Under UV, fluorescence	[6]
	O21	Au NPs, photoirradiation (NIR), fluorescence	[8]
	ROS	Under laser pulse irradiation, in cell, fluorescent marker	[2]
	ROS	Protoporphyrin IX coated, under light, fluorescence	[69]
	•OH, O2-_•	2–250 nm, X-ray and UV irradiation, fluorescence	[70]
Pt	•OH	Pt surface, H2O2, under high-voltage power supply	[71]
CoPt3	•OH, O2-_•	0.11M H2O2, ESR	[72]
FePt	ROS	In cell, PBS, fluorescence	[73]
Cu	ROS	Mercaptocarboxylic acid coated, 15 nm, fluorescence	[74]
	•OH	1mM H2O2, 1mM PBS, ESR	[75]
	O21	4–5 nm, PBS, citrate coated, by NaN3, fluorescence	[76]
Fe	•OH	O2, pH < 5, fluorescence	[77,78]
	O2-_•	O2, PBS, in cell, fluorescence	[79]
	ROS	Escherichia coli, fluorescence	[80]
	•OH	28mM H2O2, ESR	[81]
FeCo	•OH	28mM H2O2, ESR	[81]
Co	•OH	28mM H2O2, ESR	[81]
	ROS	Dose dependent, in cell, fluorescence	[82]
Ni	O21	Under UV, fluorescence	[6]
	ROS	~30 nm, in human liver cell, fluorescence	[83]
	ROS	65 nm, human lung epithelial A549 cells, fluorescence	[84]

ESR = electron spin resonance; NP = nanoparticle; PBS = phosphate-buffered saline; PVP = polyvinylpyrrolidone; ROS = reactive oxygen species; SOD = superoxide dismutase; NIR = near infrared.

3.1. ROS generation via Fenton reaction

A Fenton or Fenton-like reaction is a process that leads to the generation of hydroxyl radicals, as illustrated in reaction (2), which is best exemplified by reactions between H2O2 and Fenton-like reagents involving transition metal ions such as Fe2+ and Cu+ [59]:

Zero-valent metal NPs with relatively low redox potentials, such as Ag, Cu, and Fe NPs, can be viewed as Fenton-like NPs. They all have redox potentials less than that of H2O2/H2O (1.77 V). For example, elemental silver (ϕAg+/Ag = +0.7996 V), copper (ϕCu+/Cu = 0.52 V, and ϕCu2+/Cu = 0.34 V), and iron (ϕFe2+/Fe = −0.44 V) are thermodynamically favorable to trigger the Fenton reaction in the presence of H2O2, as displayed in reaction (3) [4]:

Therefore, a Fenton reaction is always accompanied by oxidation and dissolution of metal NPs. Dissolved metal ions such as Fe2+ and Cu+ can further promote the Fenton reaction shown in reaction (2).

He and coworkers [4] have reported the hydroxyl radicals generated from the interaction between H2O2 and Ag NPs. The formation mechanism of hydroxyl radical was suggested to take place through a Fenton reaction between Ag NPs and H2O2. Therefore, they named Ag NPs as Fenton-like NPs. Cu NPs may go through a similar process to generate hydroxyl radicals, as indicated in another study, although the authors of the study did not make such a proposal [75]. Zero-valent Fe NPs not only trigger the Fenton reaction in the presence of H2O2 to form hydroxyl radicals, but also activate dissolved oxygen to produce superoxide and H2O2, which can generate hydroxyl radicals [77-79,81]. Reactive oxygen production at neutral and alkaline pH is typically lower than that at acidic pH, due to the precipitation of metal ions and the mechanism involved in the Fenton reaction. Spherical Co and FeCo alloyed NPs were investigated by ESR spectroscopy, using DMPO as a spin trap, to generate hydroxyl radicals in the presence of 28 mM H2O2 through a Fenton-like reaction [81]. Bimetallic NPs such as CoPt and FePt were also found to produce ROS (hydroxyl radicals and superoxide) in the presence of H2O2 due to leaching of active metal ions (Fe and Co) from the particle surface, triggering a Fenton reaction [72,73].

3.2. Surface plasmon resonance enhancement

Because metal NPs contain free electrons, they can interact with an incident electromagnetic wave, resulting in the collective oscillation of electrons. When the frequency of the incident light photons equals the oscillating frequency of electrons, a resonance phenomenon, called localized surface plasmon resonance, occurs [85]. A local enhanced electromagnetic field formed on the surface of metal NPs makes the NPs useful not only in optical sensing and imaging [86], but also in enhancing the generation of ROS [2,6,8,69,70]. Zhang and coworkers [6] found that, under UV irradiation (365 nm), Ag NPs generated superoxide and hydroxyl radicals, whereas Au NPs and Ni NPs generated only singlet oxygen. They proposed that the ROS generation from Au, Ag, and Ni was primarily due to surface plasmon resonance effects. In another study, Vankayala et al [8] reported that both Ag and Au NPs can generate singlet oxygen upon photoirradiation of the surface plasmon resonance band, even at near-infrared wavelengths. Researchers characterized and quantified, using different fluorescent markers, the significant elevation in ROS generation from antibody-coated Au NPs irradiated by a few resonant femtosecond laser pulses, which resulted in a high concentration of ROS and local damage of cancer cells [2]. Au NPs can also enhance and improve the generation of ROS from photosensitizers located near the Au NP surface. Oo et al [69] have demonstrated that ROS formation by Au NPs is significantly enhanced upon irradiation with a PpIX photosensitizer because of the localized electromagnetic field of surface plasmon resonance of the illuminated Au NPs. They also found that ROS enhancement, leading to damage of breast cancer cells, is proportional to the size of Au NPs. Additionally, photo- and Auger-electron charge transfer may influence the generation of near the Au NP surface, whereas X-rays are involved in the generation of •OH [70].

4. Application of ESR for the detection of ROS generated by nanomaterials

4.1. Application of ESR spin trapping for the detection of hydroxyl radicals mediated by nanomaterials

Hydroxyl radicals are extremely reactive and can cause oxidative damage to a majority of macromolecules in biological systems [87]. A hydroxyl radical has a very short half-life (about 1 nanosecond) and a high reactivity [88], which makes its detection challenging. However, they readily react with diamagnetic nitrone spin traps, forming stable free radicals (spin adducts) that can be identified from the magnetic parameters of the ESR spectrum. We have used ESR spin trapping to study the generation of hydroxyl radicals by Ag and Au NPs during their interactions with H2O2 [4,5]. Four spin traps, DMPO, BMPO, DEPMPO, and POBN, were employed to characterize the generation of hydroxyl radicals in the presence of H2O2 and Ag NPs under acidic conditions (pH 3.6; Fig. 5). Compared with control conditions, we observed ESR spectra characteristic of adducts formed between each of the four spin traps and hydroxyl radicals, indicating that hydroxyl radicals are generated when the decomposition of hydrogen peroxide is assisted by Ag NPs under acidic conditions. Most notably, the generation of hydroxyl radical is dependent not only on the size and concentration of Ag NPs, but also on the buffer pH, as shown in Fig. 5. The pH dependence and Ag NP induction of hydroxyl radical generation were suggested to be due to the pH-dependent redox behavior of H2O2 and the ability of Ag NPs to facilitate electron transfer in different chemical environments (Fig. 5C). By employing spin-trapping ESR, we also demonstrated that Au NPs can facilitate generation of hydroxyl radicals in the presence of hydrogen peroxide [5], the pH dependence being similar to Ag NPs.

Fig. 5

(A) Demonstration of hydroxyl radicals generated by Ag NPs in the presence of hydrogen peroxide at pH 3.6 (10mM acetate buffer) using different spin traps. (B) ESR signal intensity versus buffer pH. (C) Schematic presentation of Ag NPs triggering the generation of hydroxyl radicals and oxygen controlled by pH [4]. BMPO = 5-tertbutoxycarbonyl-5-methyl-1-pyrroline N-oxide; DEPMPO = 5-diethoxyphosphoryl-5-methyl-1-pyrroline-N-oxide; DMPO = 5,5-dimethylpyrroline N-oxide; ESR = electron spin resonance; NP = nanoparticle; POBN = α-(4-pyridyl N-oxide)-N-tert-butylnitrone.

Metal oxide NPs, for example TiO2 and ZnO, are photo-catalytically active and generate ROS when photoexcited. Under some experimental conditions, particularly those found in in vitro studies, this photochemical activity can lead to cytotoxicity [9,89-91]. We employed DMPO or BMPO as a spin trap to determine whether hydroxyl radicals can be produced from irradiated TiO2 and ZnO NPs [89-91]. When TiO2 is irradiated with UV in the presence of DMPO, an ESR spectrum characteristic of the spin adduct DMPO/•OH is observed (Fig. 6A) [89]. Using BMPO as a spin trap, we further examined the generation of hydroxyl radicals for different crystalline types of TiO2. The results showed that rutile, anatase, and P25 all can produce hydroxyl radicals when irradiated, while irradiation of P25 results in the strongest signal of BMPO/OH (Fig. 6B) [9]. Similarly, ZnO NPs can generate hydroxyl radicals in a dose-dependent manner when exposed to UVA radiation, which may result in the death of human-derived keratinocytes [91]. In our collaborative work with other groups, we performed ESR spectroscopy and demonstrated that iron oxide NPs (e.g., Fe2O3 and Fe3O4 NPs) can induce the formation of hydroxyl radicals in an acidic biological microenvironment through Fenton or Fenton-like reactions [7,92].

Fig. 6

Generation of hydroxyl radicals by irradiation of TiO2 samples under UV light. ESR spectra (A) with DMPO recorded after 3 minutes of irradiation with UV radiation of 320 nm [89], and (B) with BMPO after 2 minutes of irradiation with UV 340 nm [9]. (C) Catalytic activity of GO/PP in the formation of •OH by decomposition of H2O2, using DEMPO as a spin trap. (D) A possible decomposition mechanism of hydrogen peroxide catalyzed by coronene [94]. BMPO = 5-tertbutoxycarbonyl-5-methyl-1-pyrroline N-oxide; DEPMPO = 5-diethoxyphosphoryl-5-methyl-1-pyrroline-N-oxide; DMPO = 5,5-dimethylpyrroline N-oxide; ESR =electron spin resonance; GO/PP =graphene oxide modified with PEGylated poly-L-lysine.

In collaboration with Zhao et al [93,94], we employed ESR to study the ability of carbon nanostructures, such as carbon nanotubes and graphene oxide, to generate hydroxyl radicals. Using DMPO as a spin trap, hydroxyl radicals were generated using carbon nanotubes by interaction with H2O2. This generation of hydroxyl radicals may be caused by the existence of carbonaceous and transition metallic impurities (e.g., Fe) [93]. In the study of graphene oxide (GO), functionalized GO modified with PEGylated poly-L-lysine (GO/PP) was found to catalyze the decomposition of H2O2 to form hydroxyl radicals. The hydroxyl radicals were captured by DEMPO and detected by ESR (Fig. 6C). A theoretical calculation suggests a possible decomposition mechanism of hydrogen peroxide catalyzed by coronene (Fig. 6D) [94].

4.2. Application of ESR spin trapping for the detection of superoxide anion radicals generated by nanomaterials

Among the spin traps, DMPO and BMPO are used most often for detecting superoxide anions. Harbour and Hair [95] have detected the superoxide generated from photoexcited CdS dispersions using DMPO as a spin trap. The ESR technique along with the spin trap DMPO was also used by Wang et al [96] for studying illuminated CdIn2S4 microspheres. These investigators observed the characteristic peaks of the adducts in methanol dispersion under irradiation. Zhao et al [53] have shown that use of the spin trap BMPO has advantages over DMPO as a spin trap for superoxide. This is because the BMPO/•OOH adduct is more stable and does not decompose into the corresponding hydroxyl adduct (i.e., BMPO/•OH). However, the spin adducts BMPO/•OOH and BMPO/•OH have overlapping ESR spectra, and so also the ESR spectra for DMPO/•OOH and DMPO/•OH. Therefore, it is difficult to distinguish between hydroxyl radicals and superoxide if they are generated simultaneously in one system. An examination of the quenching effect of superoxide dismutase (SOD) on superoxide or that of DMSO on hydroxyl radicals can provide additional confirmation of the existence of these free radicals. Zhao et al [97] have reported that excitation of BiOCl nanostructures with UV results in the appearance of a strong four-line ESR spectrum with splitting parameters of aN = 13.56, aH = 12.30, and aH = 0.66, which is the characteristic spectrum of BMPO/•OH adduct. Similar results were obtained for BiOCl with dominantly exposed faces 001 (BOC-001) and 010 (BOC-010). The superoxide was also captured to form the BMPO/O2−• adduct having a four-line spectrum with relative intensities of 1:1:1:1 and hyperfine splitting parameters of aN = 13.56 and aH = 12.10, which overlaps with the BMPO/•OH spectrum. To verify whether the ESR signal involved superoxide, SOD was added. After the addition of SOD, the ESR signal intensity decreased; however, no similar decrease was observed for BOC-001 and BOC-010 (Fig. 7). These observations indicated that although both •OH and O2−• were generated from irradiated BOC, •OH dominated, resulting in the unclear characteristic ESR signal for O2−• [97].

Fig. 7

ESR spectra of active oxygen radicals generated during the photocatalysis of BOC-001 and BOC-010 under UV irradiation (A) prior to and (B) after the addition of SOD [97]. BMPO = 5-tertbutoxycarbonyl-5-methyl-1-pyrroline N-oxide; BOC-001 = BiOCl with dominantly exposed face 001; BOC-010 = BiOCl with dominantly exposed face 010; ESR = electron spin resonance; SOD = superoxide dismutase.

4.3. Application of ESR spin trapping for the detection of singlet oxygen generated by nanomaterials

Singlet O2 is a very important ROS involved in peroxidation of olefins, photobiological cytotoxicity, and, importantly, clinical photodynamic therapy for killing cancer cells. Upon irradiation, a variety of nanomaterials, including metal NPs (Au, Ag, Ni, etc.) [6,8], semiconductor NPs (Si, TiO2, ZnO, etc.) [9,41,98], and fullerenes [39,40], can efficiently generate singlet oxygen. Singlet oxygen can be formed by photoexcitation of metal NPs due to their surface plasmon resonance properties. For example, Au nanorods generate singlet oxygen via photoexcitation in the near-infrared region, which may potentially lead to their use in photodynamic and photothermal therapies for cancer treatment [8]. Additionally, semiconductor NPs such as TiO2, ZnO, etc. are well-known photocatalysts. Because of their electronic structure, these materials are capable of generating singlet oxygen on photoexcitation. As discussed in Part 2.2, TEMP and 4-oxo-TEMP are two typical spin traps used for the detection of singlet oxygen in ESR. We have examined the generation of singlet oxygen from irradiated TiO2 by the ESR trapping technique using TEMP [9]. Irradiation of different nanoscale TiO2 samples containing 20 mM TEMP resulted in an ESR spectrum consisting of three lines with equal intensities (aN = 16.0 G), which is typical of nitroxide radicals (Fig. 8, inset). The hyperfine splitting constant and g factor are characteristic of TEMPONE. The ESR intensity of the TEMPONE signal increased as a function of time during irradiation of different nano-TiO2 samples. A control solution containing only TEMP did not lead to an increase in the ESR signal. For P25 nano-TiO2, the addition of a singlet oxygen quencher, NaN3, caused a significant reduction in the rate of increase of the electron paramagnetic resonance signal (Fig. 8, curve E). The rates of production of singlet oxygen for the different species followed the trend P25 > A25 > A325 > R100 nano-TiO2 (Fig. 8).

Fig. 8

Generation of singlet oxygen by photoexcitation of TiO2 samples under UVA light in time- and crystal type-dependent manners. ESR spectra were recorded at room temperature. Samples containing 20mM TEMP and 0.1 mg/mL P25 (curve A), 0.1 mg/mL A25 (curve B), 0.1 mg/mL A325 (curve C), and 0.1 mg/mL R100 (curve D), and that containing 10 mM NaN3 and 0.1 mg/mL P25 (curve E) were irradiated with UVA light at 340 nm. Inset: ESR signal of TEMPONE (aN = 16.0 G) [9]. ESR = electron spin resonance; TEMP = 2,2,6,6-tetramethylpiperidine.

Zhao et al [41] studied the encapsulation of silicon phthalocyanine 4 (Pc4) in silica NPs to enhance photodynamic efficacy toward melanoma cells. Using the spin trap TEMP, they showed that photoexcited Pc4 encapsulated in silica NPs, with a particle size in the range of 25–30 nm, generated singlet oxygen. Intensity of the ESR signals from TEMPONE was enhanced progressively when the irradiation time was extended. These results provide direct evidence that the photoexcitation of Pc4 encapsulated in silica NPs with visible light (>550 nm) generates singlet oxygen and that the quantity of singlet oxygen formed is dependent on the dose of administered light. In addition to Pc4 and silica NPs, fullerenes were also studied. The water-soluble fullerene derivative γ-cyclodextrin bicapped C60 [(γ-CyD)2/C60, CDF0] was found to be a highly efficient photosensitizer for the generation of singlet oxygen. In a study assessing its potential phototoxicity in human lens epithelial cells (HLE B-3) in vitro, using the same ESR/TEMP trapping technique, Zhao et al [40] found that (γ-CyD)2/C60 (CDF0) can produce singlet oxygen efficiently. A solution of (γ-CyD)2/C60 (CDF0) showed the highest rate of singlet oxygen production; the rate decreased with increasing aggregation, with no production by the fully aggregated sample after 150 minutes of heating (CDF150). They concluded that singlet oxygen is an important intermediate in the phototoxicity of monomeric (γ-CyD)2/fullerene.

4.4. Application of ESR spin label oximetry for studying lipid peroxidation by nanomaterials

In the detection of O2, ESR oximetry has a number of advantages. For example, ESR oximetry does not lead to consumption of O2 during measurement and it is nondestructive. In addition, ESR oximetry has less interference with small molecules and requires less sample volume while maintaining high sensitivity [57]. Chemically, lipid peroxidation can be caused by O2, light, ROS, and other free radicals. Importantly, lipid peroxidation is associated with the consumption of O2. Therefore, ESR spin label oximetry is very suitable for sensitive detection of lipid oxidation by measuring O2 consumption. In addition, ESR oximetry can also provide dynamic information on the rate of lipid oxidation; this can be obtained by assessing the variation in O2 uptake with time, calculated from the line widths of the spin label.

We have used ESR oximetry in conjunction with the spin label CTPO to monitor the effect of Au NPs in triggering the production of oxygen under biologically relevant conditions [5]. Resolution of the superhyperfine structure of the low-field line of the ESR spectrum of CTPO strongly depends on the O2 concentration in the sample solution. An increase in the oxygen concentration results in a progressive reduction of the superhyperfine structure, with the eventual loss of the hyperfine structure. As seen in Fig. 9, the structure of the ESR signal of spin label CTPO was affected by various conditions. An ESR signal with a typical sharp superhyperfine structure and a high intensity of hyperfine structure was observed in the control sample without catalysts, indicating a low concentration of oxygen. However, when Au NPs were added, the superhyperfine structures diminished along with a corresponding decreased signal intensity that suggests the formation of O2. The superhyperfine splitting of spin label clearly diminished with increasing pH, especially in samples with pH ranging from 6.0 to 11.

Fig. 9

Generation of O2 induced by Au NPs under different experimental conditions. ESR spectra of 0.1mM CTPO in 10 mM buffers having different pH, in the presence of 0.5 mM H2O2 and 0.1 mg/mL Au NPs (particle size 10 nm). Sample solutions were aerated with nitrogen for 15 minutes prior to mixing. ESR spectra were collected after 6 minutes of incubation [5]. CTPO = 3-carbamoyl-2,2,5,5-tetra-methyl-3-pyrroline-1-yloxyl; ESR = electron spin resonance; NP = nanoparticle; PVP = polyvinylpyrrolidone.

It is well known that ROS can induce time-dependent peroxidation of the polyunsaturated lipids in plasma membrane. Yin et al [9] studied lipid peroxidation by UVA irradiation of nano-TiO2 using ESR oximetry. Consumption of oxygen associated with lipid peroxidation was measured from the time-dependent narrowing of the ESR signal for the spin probe 15N-PDT (Fig. 10). The narrowing of the ESR signal was accompanied by an increase in its peak height within the scan range. The final ESR signal intensities of R100, A325, A25, and P25 nano-TiO2 samples were approximately 7.5%, 11.7%, 16.2%, and 26.3%, respectively, higher than the control (Fig. 10). The progressive increases in peak-to-peak signal intensity along with narrowing of the line width in each panel were due to time-dependent oxygen consumption associated with lipid peroxidation. Fig. 10F presents the data as a decrease in oxygen concentration, reflecting the variations in the slope (oxygen concentration vs. time) with different nano-TiO2 species, which is in the following order: P25 > A25 > A325 > R100.

Fig. 10

Effect of TiO2 samples on lipid peroxidation in liposomes. Oxygen consumption was measured in a closed chamber using liposome suspensions and the spin label 15N-PDT. The liposome sample contained 30 mg/mL Egg PC and 0.1mM 15N-PDT spin label mixed with no TiO2 (curve “a” in Fig. 10F), 0.03 mg/mL of R100 (curve “b” in Fig. 10F), 0.03 mg/mL of A325 (curve “c” in Fig. 10F), 0.03 mg/mL of A25 (curve “d” in Fig. 10F), and 0.03 mg/mL of P25 (curve “e” in Fig. 10F). Lipid peroxidation was initiated by UV (340 nm) irradiation [9]. 15N-PDT = 4-oxo-2,2,6,6-tetramethyl piperidine-d16-1-15N-oxyl.

5. Conclusions

The production of ROS induced by nanomaterials is a double-edged sword, bringing not only the benefits of efficient nanomaterials for therapeutic treatment of diseases, but also possible health and environmental risks associated with them. Therefore, it is important to identify ROS for developing nanomaterials for specific applications and understanding risks associated with their use. The use of ESR techniques to study ROS generation mediated by nanomaterials has several advantages compared to other techniques. Most importantly, ESR provides a more direct and chemically specific method for detecting ROS formation and identifying free radical species.

Because nanomaterials can affect cellular function through the production of ROS, ESR is an important technique used to study the free radical mechanisms of nanomaterial toxicity. In fact, the ability of nanomaterials to facilitate electron transfer, and thereby promote ROS generation, may be a fundamental property of these materials. It is still not clear whether and how the ROS production is associated with the physicochemical characteristics in terms of mechanisms and activity. For example, what factors cause the different type of ROS generated by different kinds of nanomaterials? Is there any dependence on the size, shape, or crystal facet of nanomaterials in the generation of ROS? In addition, how does ROS contribute to the toxicity of nanomaterials? To answer these questions, one requires to have not only a clear understanding of the mechanism of ROS generation in different types of nanomaterials, but also the knowledge of standard nanomaterials with well-controlled size, shape, composition, and surface states to compare their abilities in generating ROS.