**Meet the editor**

Dr. Mahmood Aliofkhazraei works in the Corrosion and Surface Engineering Group at the Tarbiat Modares University, Iran. He is the head of Aliofkhazraei research group (www.aliofkhazraei.com). Dr. Aliofkhazraei has received several honors, including the Khwarizmi award and the best young nanotechnologist award of Iran. He is a member of the National Association of Surface Sciences,

Iranian Corrosion Association, and National Elite Foundation of Iran. His research focuses on materials science, nanotechnology and its use in surface and corrosion science.

### Contents

**Preface XI**


Chapter 6 **The Development of Smart, Multi-Responsive Core@Shell Composite Nanoparticles 103** Bo Sang Kim and T. Randall Lee

### Preface

During the past years, scientists have achieved significant success in nano science and technol‐ ogy. Nanotechnology is a branch of science which deals with fine structures and the materials with very small dimensions – less than 100 nm. Measurement unit of nano has been extracted from nano prefix, which is a Greek word meaning extremely fine. One nano (10 -9 m) is the length equivalent to 5 silicon atoms or 10 hydrogen atoms aligned side by side. In perspective, note the following examples: Hydrogen atom is about 0.1 nm; a virus is about 100 nm; diame‐ ter of a red blood cell is 7000 nm; and diameter of a human hair is 10000nm. Nanotechnology is a field of applied sciences which is focused on design, production, detection, and employing the nano-size materials, pieces, and equipment. Advances in nanotechnology lead to improve‐ ment of tools and equipment as well as their application in human life. "Nano science" is study of the phenomena emerged by atomic or molecular materials with the size of several nanome‐ ters to less than 100 nm. In the chemistry this size involves the range of colloids, micelles, polymer molecules, and structures such as very large molecules or dense accumulation of the molecules. In physics of electrical engineering, the nanoscience is strongly related to quantum behavior or electrons behavior in structures with nano sizes. In biology and biochemistry, also, interesting cellular components and molecular structures such as DNA, RNA, and intercellu‐ lar components are considered as nanostructures.

This book collects new developments about nanoparticles. I like to express my gratitude to all of the contributors for their high quality manuscripts. I hope open access format of this book will help all researchers and that they will benefit from this collection.

> **Dr. Mahmood Aliofkhazraei** Tarbiat Modares University Iran www.aliofkhazraei.com

## **Nanoparticle Formation by Laser Ablation and by Spark Discharges — Properties, Mechanisms, and Control Possibilities**

Andrey Voloshko and Tatiana E. Itina

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/61303

### **Abstract**

Laser ablation (LA) and spark discharge (SD) techniques are commonly used for nanoparticle (NP) formation. The produced NPs have found numerous applications in such areas as electronics, biomedicine, textile production, etc. Previous studies pro‐ vide us information about the amount of NPs, their size distribution, and possible ap‐ plications. On one hand, the main advantage of the LA method is in the possibilities of changing laser parameters and background conditions and to ablate materials with complicated stoichiometry. On the other hand, the major advantage of the SD techni‐ que is in the possibility of using several facilities in parallel to increase the yield of nanoparticles. To optimize these processes, we consider different stages involved and analyze the resulting plasma and nanoparticle (NP) parameters. Based on the per‐ formed calculations, we analyze nanoparticle properties, such as mean size and mean density. The performed analysis (shows how the experimental conditions are connect‐ ed with the resulted nanoparticle characteristics in agreement with several previous experiments. Cylindrical plasma column expansion and return are shown to govern primary nanoparticle formation in spark discharge, whereas hemispherical shock de‐ scribes quite well this process for nanosecond laser ablation at atmospheric pressure. In addition, spark discharge leads to the oscillations in plasma properties, whereas monotonous behavior is characteristic for nanosecond laser ablation. Despite the dif‐ ference in plasma density and time evolutions calculated for both phenomena, after well-defined delays, similar critical nuclei have been shown to be formed by both techniques. This result is attributed to the fact that whereas larger evaporation rate is typical for nanosecond laser ablation, a mixture of vapor and background gas deter‐ mines the supersaturation in the case of spark.

**Keywords:** Nanoparticles, laser ablation, plasma, spark discharge, synthesis, model‐ ing, size distribution, nucleation, aggregation

© 2015 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

### **1. Introduction**

Modern nanotechnology includes several promising areas such as nano-optics, nano-photon‐ ics, nanochemistry, nanobiology, and nanomedicine. During the past decade, we have witnessed a tremendous growth of nanoparticle applications that require particles of different materials with different size, dispersion, shape, and morphology. As a result, the development of new nanoparticle synthesis methods is particularly important. Among the rapidly im‐ merged techniques, plasma-based synthesis has a number of advantages being both rather simple and allowing unique and well-controlled formation of nanoparticles.

In particular, plasmas created by both pulsed laser ablation and by spark discharges can be used for nanoparticle synthesis. That is why these two techniques have attracted particular attention and resulted in numerous experimental and theoretical investigations. On one hand, the main advantage of laser ablation method, as was demonstrated in these studies, is the possibility to preserve target stoichiometry. On the other hand, spark discharge allows one to produce a very large amount of nanoparticles by using parallel multidischarge set-up. However, the main physical mechanisms involved in these processes stay partly puzzling. That is why additional studies based on a detailed comparison of both methods are required for the determination of their main similarities and differences.

Starting from the early 1990s, laser ablation (LA) has been intensively studied first for long laser pulses and then for much shorter ones [1-2]. A number of experiments revealed that laser interactions with solid targets lead to the formation of nanoparticles. Furthermore, if femto‐ second laser is used, an explosive ejection of a mixture of clusters and atoms was both theoretically predicted and experimentally observed [3]. It was found that the produced nanoparticles demonstrated either plasmonic or photoluminescent properties, as well as a capacity of field amplification. These properties are particularly interesting biomedical applications, such as imaging and photodynamic therapy. It should be noted that absence of incompatibility with biological tissues is crucial for further development of most of the applications where nanoparticles are used in vivo. However, toxicity is hard to avoid in traditional chemical methods. In addition, the stability of these nanoparticles is still not high enough. It was demonstrated, fortunately, that nanoparticles produced by laser ablation are better suitable for biomedical applications, in particular, when they are produced in liquids [4]. This advantage made LA a unique tool for nanoparticle synthesis.

In order to elucidate the physical mechanisms of LA, many analytical and numerical investi‐ gations were performed [5 -11]. In vacuum, self-similar adiabatic models with condensation were proposed [10-11]. In the presence of a gas, only either very low or high background pressure was used in most of the models for simplicity. Shock waves were shown to be produced during the plume expansion in a high-pressured background gas [3]. In this case, a system of Navier-Stocks equations well describes the first 1–2 μs of the plasma plume expan‐ sion. It should be noted that such models are invalid at the later stages. To solve this issue, hydrodynamic calculations were switched to the direct Monte Carlo simulations where no such hypothesis is used at 1–2 μs after the laser pulse [11, 12]. Recently, such approaches as molecular dynamics (MD), hydrodynamics (HD), and combinations with the direct simulation Monte Carlo method (DSMC) were proposed for picosecond and femtosecond laser interac‐ tions [13-14]. The initial stage of laser ablation process can be examined by using either hydrodynamic models or atomistic simulation. On one hand, the main advantage of numerical hydrodynamics is in the calculation rapidity and in the possibility to reach rather larger scales [10, 13]. Atomistic simulations, on the other hand, are not based on equilibrium assumptions and can more easily provide size distributions of nanoparticles. In particular, two-temperature molecular dynamics simulations (TTM-MD) were performed for femtosecond laser ablation of metals [14, 15].

In the presence of a sufficiently high-pressured background environment, such as atmospheric pressured gas or a liquid, diffusion-driven nucleation and aggregation processes start playing an important role (Figure 1) at longer delays [16].

**Figure 1.** Nanoparticle formation and growth schematics.

**1. Introduction**

2 Nanoparticles Technology

Modern nanotechnology includes several promising areas such as nano-optics, nano-photon‐ ics, nanochemistry, nanobiology, and nanomedicine. During the past decade, we have witnessed a tremendous growth of nanoparticle applications that require particles of different materials with different size, dispersion, shape, and morphology. As a result, the development of new nanoparticle synthesis methods is particularly important. Among the rapidly im‐ merged techniques, plasma-based synthesis has a number of advantages being both rather

In particular, plasmas created by both pulsed laser ablation and by spark discharges can be used for nanoparticle synthesis. That is why these two techniques have attracted particular attention and resulted in numerous experimental and theoretical investigations. On one hand, the main advantage of laser ablation method, as was demonstrated in these studies, is the possibility to preserve target stoichiometry. On the other hand, spark discharge allows one to produce a very large amount of nanoparticles by using parallel multidischarge set-up. However, the main physical mechanisms involved in these processes stay partly puzzling. That is why additional studies based on a detailed comparison of both methods are required

Starting from the early 1990s, laser ablation (LA) has been intensively studied first for long laser pulses and then for much shorter ones [1-2]. A number of experiments revealed that laser interactions with solid targets lead to the formation of nanoparticles. Furthermore, if femto‐ second laser is used, an explosive ejection of a mixture of clusters and atoms was both theoretically predicted and experimentally observed [3]. It was found that the produced nanoparticles demonstrated either plasmonic or photoluminescent properties, as well as a capacity of field amplification. These properties are particularly interesting biomedical applications, such as imaging and photodynamic therapy. It should be noted that absence of incompatibility with biological tissues is crucial for further development of most of the applications where nanoparticles are used in vivo. However, toxicity is hard to avoid in traditional chemical methods. In addition, the stability of these nanoparticles is still not high enough. It was demonstrated, fortunately, that nanoparticles produced by laser ablation are better suitable for biomedical applications, in particular, when they are produced in liquids [4].

In order to elucidate the physical mechanisms of LA, many analytical and numerical investi‐ gations were performed [5 -11]. In vacuum, self-similar adiabatic models with condensation were proposed [10-11]. In the presence of a gas, only either very low or high background pressure was used in most of the models for simplicity. Shock waves were shown to be produced during the plume expansion in a high-pressured background gas [3]. In this case, a system of Navier-Stocks equations well describes the first 1–2 μs of the plasma plume expan‐ sion. It should be noted that such models are invalid at the later stages. To solve this issue, hydrodynamic calculations were switched to the direct Monte Carlo simulations where no such hypothesis is used at 1–2 μs after the laser pulse [11, 12]. Recently, such approaches as molecular dynamics (MD), hydrodynamics (HD), and combinations with the direct simulation

simple and allowing unique and well-controlled formation of nanoparticles.

for the determination of their main similarities and differences.

This advantage made LA a unique tool for nanoparticle synthesis.

However, despite a growing interest in LA, some of its basic mechanisms remain not enough understood. The challenge is that ablation processes strongly depend on the ensemble of such parameters as temporal pulse width and shape, on laser wavelength, on the size of the laser spot, laser intensity, repetition rate, as well on the target material and background conditions. [17]

In spark discharges (SD), a typical set-up consists of two electrodes connected to a charged capacitance [16]. When high enough voltage *V0* is applied, a so-called streamer is formed first. Once it reaches the opposite electrode, plasma breakdown takes place, followed by the streamer transition to an expanding plasma column. In this column, Joule heating of both plasma and electrodes take place. In addition, electrodes are bombarded by energetic ions that induce sputtering, which can be considered to be similar to laser ablation. If background gas is present, rapid thermalization of the sputtered material leads to primary nanoparticle formation that can then grow by collisions and form larger particles or aggregates.

Numerical modeling of spark discharge consists of several steps with rather different time scales [16]: (i) streamer formation and propagation between electrodes; (ii) streamer-to-spark transition; (iii) gas heating and cylindrical expansion, (iv) electrode evaporation and erosion; (v) nanoparticles formation. Nanoparticle formation, here as in the case of laser ablation in the presence of a gas or a liquid, includes nucleation and collisional growth (Figure 1).

This work is aimed at the better understanding of the mechanisms involved in nanoparticle formation by laser ablation and by spark discharge. First, laser ablation is considered by using both atomistic and hydrodynamic numerical methods. In particular, mechanisms of nanopar‐ ticle formation and the corresponding conditions are analyzed. Then, attention is focused on the role of the background environment and its role in nanoparticle formation. These results are used to explain several recent experimental results.

Second, spark discharge is investigated. Plasma properties and conditions required for nanoparticle formation are examined. Finally, we compare laser ablation and spark discharge as promising methods of nanoparticle formation.

### **2. Laser ablation**

To examine ultra-short, laser-ablated plume dynamics and nanoparticle evolution under realistic experimental conditions and to account for the fact that the ablated plume contains several components, DSMC calculations of the plume dynamics are first performed in the presence of an inert background gas (Ar) with pressure *P* = 300 Pa. The initial conditions are set based on the parameterization of the MD results obtained at a delay of 200 ps after the beginning of the laser pulse (100 fs, 800 nm) [16].

Figure 2 shows separately the density of atoms and clusters for two different delays after the laser pulse. Here, larger clusters were initially at the back of the plume. The obtained results demonstrate that plume front starts experiencing a pronounced deceleration and practically stops at both plume- and gas-dependent delay (here, ~10μs). Theoretically, the initial expan‐ sion stage is described by a so-called blast-wave (or, shock-wave) model when shock waves are degenerated.

The corresponding nanoparticle size distributions are presented in Figure 3. One can see that after a sufficient delay, a peaked distribution appears instead of a decreasing function. This effect can be explained by collisional growth that is described by the general rate equation having typically log-normal solutions. The amount of sufficiently large nanoparticles formed at such short delays is rather small and cannot explain the finally observed size distributions.

Longer stage includes plume mixing with the background followed by the rapid thermaliza‐ tion and a much more enhanced particle formation. Then, plume species are thermalized and a diffusion-driven regime enters into play.

### **3. Spark discharge**

In our model developed for spark discharge (SD), the following stages are considered: (i) streamer formation and propagation between electrodes; (ii) streamer-to-spark transition; (iii) gas heating and cylindrical expansion; (iv) electrode evaporation and erosion; (v) nanoparticles formation [16].

Nanoparticle Formation by Laser Ablation and by Spark Discharges — Properties, Mechanisms, and... http://dx.doi.org/10.5772/61303 5

This work is aimed at the better understanding of the mechanisms involved in nanoparticle formation by laser ablation and by spark discharge. First, laser ablation is considered by using both atomistic and hydrodynamic numerical methods. In particular, mechanisms of nanopar‐ ticle formation and the corresponding conditions are analyzed. Then, attention is focused on the role of the background environment and its role in nanoparticle formation. These results

Second, spark discharge is investigated. Plasma properties and conditions required for nanoparticle formation are examined. Finally, we compare laser ablation and spark discharge

To examine ultra-short, laser-ablated plume dynamics and nanoparticle evolution under realistic experimental conditions and to account for the fact that the ablated plume contains several components, DSMC calculations of the plume dynamics are first performed in the presence of an inert background gas (Ar) with pressure *P* = 300 Pa. The initial conditions are set based on the parameterization of the MD results obtained at a delay of 200 ps after the

Figure 2 shows separately the density of atoms and clusters for two different delays after the laser pulse. Here, larger clusters were initially at the back of the plume. The obtained results demonstrate that plume front starts experiencing a pronounced deceleration and practically stops at both plume- and gas-dependent delay (here, ~10μs). Theoretically, the initial expan‐ sion stage is described by a so-called blast-wave (or, shock-wave) model when shock waves

The corresponding nanoparticle size distributions are presented in Figure 3. One can see that after a sufficient delay, a peaked distribution appears instead of a decreasing function. This effect can be explained by collisional growth that is described by the general rate equation having typically log-normal solutions. The amount of sufficiently large nanoparticles formed at such short delays is rather small and cannot explain the finally observed size distributions. Longer stage includes plume mixing with the background followed by the rapid thermaliza‐ tion and a much more enhanced particle formation. Then, plume species are thermalized and

In our model developed for spark discharge (SD), the following stages are considered: (i) streamer formation and propagation between electrodes; (ii) streamer-to-spark transition; (iii) gas heating and cylindrical expansion; (iv) electrode evaporation and erosion; (v) nanoparticles

are used to explain several recent experimental results.

as promising methods of nanoparticle formation.

beginning of the laser pulse (100 fs, 800 nm) [16].

a diffusion-driven regime enters into play.

**2. Laser ablation**

4 Nanoparticles Technology

are degenerated.

**3. Spark discharge**

formation [16].

**Figure 2.** Calculated plume dynamics for Ni expansion in Ar gas at 300 Pa, (a) density snapshot for atoms a*tt* = 0.55 μs, (b) the same for clusters at *t* = 0.55 μs; (c) the same for atoms at *t* = 10 μs, (d) the same for clusters a*t t* = 10 μs.

Streamer formation is described numerically by using a system of drift-diffusion equations together with Poisson equation for electric potential with a particularly chosen set of boundary conditions. When streamer reaches the opposite electrode, electron emission increases dramatically, so that the streamer is transformed in a conductive plasma column. The oscilla‐ tions of the electric charge Q in the corresponding circuit with the total resistance *R<sup>Σ</sup>* is described by the Kirchhoff's voltage law.

The oscillating behavior of the discharge is presented in Figure 4, defining the properties of the following plasma column. According to this solution, both electrodes play a role at different delays leading to both evaporation and erosion of the electrodes. When polarity switches, a crater is formed at the surface of one of the electrodes due to both evaporation and erosion.

The erosion flux *j surf <sup>Σ</sup>* is formed due to two main processes: (i) thermal evaporation caused by Joule heating, and (ii) sputtering due to ion bombardment. Similarly to nanosecond laser ablation, evaporation flux *j surf <sup>T</sup>* is described by the Hertz-Knudsen equation, where surface temperature is calculated as follows [16]

**Figure 3.** Size distributions calculated by using MD-DSMC model in the presence of 300 Pa of Ar at different time de‐ lays.

**Figure 4.** Typical time evolutions of voltage and electric current during a "single" spark event obtained in N2 for *C* = 8 nF; *L* = 0.77 μH; *RΣ* = 1 Ω.

$$\begin{aligned} \left. \varepsilon \rho\_s \frac{\partial T\_s}{\partial t} = \frac{\overline{f}\_s^{\prime 2}}{\sigma\_s} + \rho\_s \overline{\nabla} \cdot \left( \underline{\chi}\_s \overline{\nabla} \left( kT\_s \right) \right) \right| \\ \left. \frac{\partial T\_s}{\partial z} \right|\_{z=0} &= 0, \left. T\_s \right|\_{z=L} = \left. 300 K\_r \left. \frac{\partial T\_s}{\partial r} \right|\_{r=0} = 0, \left. T\_s \right|\_{r=r\_{\text{max}}} = 300 K\_r \\ \left. \dot{T}\_{surf}^{\overline{\Gamma}} = \frac{\alpha \left( P\_{\text{eq}} - P\_{\text{surf}} \right)}{\sqrt{2 \pi mkT\_s}} \right| \end{aligned} \tag{1}$$

where *s* corresponds to the cathode material (solid), *c* is the specific heat of cathode material, *ρs* is the density, *Ts* is the temperature, *σs* is the electric conductivity, *χ<sup>s</sup>* is the thermal diffusivity coefficient, *k* is the Boltzmann constant, *α* is the sticking coefficient for vapor molecules onto the surface, *Peq* is the equilibrium vapor pressure, and *Psurf* is the hydrostatic pressure of gas applied on the surface.

The sputtering yield Y *Y* and flux *j surf sput* is given by

( ( ))

¶ ¶ == = = ¶ ¶

,

**Figure 4.** Typical time evolutions of voltage and electric current during a "single" spark event obtained in N2 for *C* = 8

**Figure 3.** Size distributions calculated by using MD-DSMC model in the presence of 300 Pa of Ar at different time de‐

0, 300 , 0, 300 ,

*max*

(1)

= =

*s s z L r r*

*T T TK T K*

( )

*P P*

2


*<sup>j</sup> mkT*

*T eq surf*

p

a

0 0

= =

*z r*

*s*

2

s

*s e*

 rc

*T j <sup>c</sup> kT*

*s s ss s*

= + Ñ× Ñ

*s s*

*z r*

<sup>r</sup> r r

r

nF; *L* = 0.77 μH; *RΣ* = 1 Ω.

lays.

6 Nanoparticles Technology

*surf*

¶

¶

*t*

$$\begin{aligned} Y &= \frac{3\alpha\_{surf}}{4\pi^2} \frac{4mM}{\left(m+M\right)^2} \frac{E\_\circ}{\mathcal{U}\_{surf}} \\ \dot{j}\_{surf}^{sput} &= \dot{j}\_\circ Y, \ \dot{j}\_{surf}^{\Sigma} = \dot{j}\_{surf}^{\Gamma} + \dot{j}\_{surf}^{sput} \end{aligned} \tag{2}$$

where *αsurf* is a factor function of *m/M*, *m* and *M* are the atomic weights of cathode material and incident particles, respectively, *E+* is the bombarding energy, *Usurf* is the surface-binding energy, and *j+* is the flux density of bombarding ions. The solution of Eqs (1–2) yields cathode erosion flow as a function of time. At the same time, plasma column also gains energy by Joule heating.

Once the amount of the ejected material is calculated, plasma dynamics is modeled by using Navier-Stokes equations [17]. The corresponding equations contain, in particular, Joule heating term, which determines plasma heating occurring mostly near its axis, where plasma pressure initially arises to several atmospheres. Figure 5 shows typical axial temperature evolution. After a delay of 0.5 μs, which corresponds to plasma expansion, pressure drops back to the values around the atmospheric pressure. Plasma temperature remains high during all the discharging process and drops below electrode boiling point only after 0.1–0.5 ms. During cooling, gas density increases. Lager density and smaller temperature provide conditions required for nanoparticle formation.

**Figure 5.** Typical time evolutions of the plasma temperature obtained for Ar for gap of 1 mm, *R* = 1 Ω, *C* = 8 nF, and *L* = 0.77 μH.

### **4. Spark discharge vs nanosecond laser ablation**

Typically, laser energy absorption leads to the target heating and thermal evaporation in the nanosecond laser ablation of metals. Plasma expansion stage is much longer than the evapo‐ ration stage, on the order of ~10 laser pulse temporal widths (~300 ns). Here, plasma electrons gain energy from laser radiation by inverse Bremsstrahlung effect, so that ionization takes place. Then, a so-called blast wave model describes hemispherical expansion as follows [16]:

$$\mathcal{R}\_v = ^\circ \xi^\circ \left(\frac{2E\_0}{\rho\_v}\right)^{1/5} t^{2/5} \,\,\,\,\,\tag{3}$$

$$\xi = \left\{ \frac{75\left(\gamma - 1\right)\left(\gamma + 1\right)^2}{16\,\mathrm{°}\,\pi^{\mathrm{o}}\left(3\gamma - 1\right)} \right\}^{1/5} \text{ \AA} \tag{4}$$

$$T\_v = T\_0 \left(\frac{\mathcal{R}\_0}{\mathcal{R}\_v}\right)^{3(\gamma - 1)},\tag{5}$$

where *R0* is the shock wave position, *ξ* is a constant depending on the plume specific heat capacity, *E0* is the initial internal energy of vapor plume, *T0* is the initial temperature, and *γ* is the adiabatic coefficient. This model is valid, if the external shock wave is present and if the mass of the background gas surrounding the shock wave is larger than the mass of the ablated material. During the expansion, both plume temperature and density rapidly decay. As a result, the condition of supersaturation is realized at a certain delay leading to the nanoparticle formation.

Time evolutions of plasma properties are obtained in [16]. For spark discharge, when gas density decreases, plasma resistivity drops down, thus preventing further energy absorption from the electric current. During the expansion, plasma cools down and gas goes back to the axis. These processes are repeated when the energy absorption is high enough for efficient expansion.

In the case of nanosecond laser ablation, laser energy absorption by the plume takes place. As a result, the ejected plasma is hot, but the energy input is limited in time by laser pulse duration. After the end of the laser pulse, the ablated plume expands and cools down. The critical nuclei sizes are calculated as a function of time (Figure 6).

The obtained results clearly demonstrate that, despite rather different plasma sources, the behavior of particle formation in spark discharge appears to be very similar to the one in laser ablation. The difference in time delays corresponds to the transition of the system to the supersaturated state. Primary NPs formed by nucleation can then evolve due to evaporation, condensation, and/or growth.

Nanoparticle Formation by Laser Ablation and by Spark Discharges — Properties, Mechanisms, and... http://dx.doi.org/10.5772/61303 9

**Figure 6.** Typical time evolution of primary critical clusters size formed by spark discharge and by laser ablation.

Diffusion-driven nucleation leads to the formation of nucleus, whose size is controlled by the free energy as follows [18]:

$$
\Delta G\left(n, c\right) = -nkT\ln\left(c\left/c\_{eq}\right) + 4\pi a^2 n^{2/3} \sigma\_\*\tag{6}
$$

where *k* is the Boltzmann constant; *T* is the temperature in Kelvins; *a* is the effective radius; *ceq* is the equilibrium concentration of atoms/monomers; *n* is the number of atoms/monomers in the nuclei; and *σ* is the effective surface tension. The peak of the nucleation barrier corresponds to the critical cluster size

$$m\_c = \left[\frac{8\pi a^2 \sigma}{3kT \ln\left(c/c\_{eq}\right)}\right]^3. \tag{7}$$

The production rate of the supercritical nuclei is given by

**4. Spark discharge vs nanosecond laser ablation**

Typically, laser energy absorption leads to the target heating and thermal evaporation in the nanosecond laser ablation of metals. Plasma expansion stage is much longer than the evapo‐ ration stage, on the order of ~10 laser pulse temporal widths (~300 ns). Here, plasma electrons gain energy from laser radiation by inverse Bremsstrahlung effect, so that ionization takes place. Then, a so-called blast wave model describes hemispherical expansion as follows [16]:

> 1/5 <sup>2</sup> <sup>0</sup> 2/5 ,

> > 1/5 <sup>2</sup>

*<sup>E</sup> R t* (3)

*<sup>R</sup>* (5)

(4)

x

g

p g

æ ö <sup>=</sup> ç ÷ ç ÷ è ø *<sup>v</sup> v*

*<sup>R</sup> T T*

x

sizes are calculated as a function of time (Figure 6).

condensation, and/or growth.

formation.

8 Nanoparticles Technology

expansion.

r

( )( ) ( )

ì ü ï ï - + <sup>=</sup> í ý °° - ï ï î þ

75 1 1 , 16 3 1

3 1 ( ) 0 <sup>0</sup> , g-

where *R0* is the shock wave position, *ξ* is a constant depending on the plume specific heat capacity, *E0* is the initial internal energy of vapor plume, *T0* is the initial temperature, and *γ* is the adiabatic coefficient. This model is valid, if the external shock wave is present and if the mass of the background gas surrounding the shock wave is larger than the mass of the ablated material. During the expansion, both plume temperature and density rapidly decay. As a result, the condition of supersaturation is realized at a certain delay leading to the nanoparticle

Time evolutions of plasma properties are obtained in [16]. For spark discharge, when gas density decreases, plasma resistivity drops down, thus preventing further energy absorption from the electric current. During the expansion, plasma cools down and gas goes back to the axis. These processes are repeated when the energy absorption is high enough for efficient

In the case of nanosecond laser ablation, laser energy absorption by the plume takes place. As a result, the ejected plasma is hot, but the energy input is limited in time by laser pulse duration. After the end of the laser pulse, the ablated plume expands and cools down. The critical nuclei

The obtained results clearly demonstrate that, despite rather different plasma sources, the behavior of particle formation in spark discharge appears to be very similar to the one in laser ablation. The difference in time delays corresponds to the transition of the system to the supersaturated state. Primary NPs formed by nucleation can then evolve due to evaporation,

 g

æ ö =° °ç ÷ ç ÷ è ø *<sup>v</sup> v*

$$\nu\left(t\right) = \mathcal{K}\_c c^2 \exp\left[\frac{-\Delta G\left(n\_{c'}c\right)}{kT}\right].\tag{8}$$

As a result, narrow size distributions are produced. The formed particles can collide and aggregate. These collisional processes are described by simplified Smoluchowski equations [18]. Laser-induced fragmentation is neglected here.

Several calculations are performed to study time-evolution of the size distribution for multiple pulse cases. Because saturation is rather high, monomer radius here is as small as *a* = 1.59–10 and the radius of critical nuclei is recalculated at all the time-steps.

**Figure 7.** Calculated size distribution obtained for 10, 100, 1000, and 2000 pulses. Here, laser frequency is 1 kHz, gold solution in water is considered with *a* = 1.59–10 m.

The obtained results (Figure 7) show that when several pulses are applied, these small nuclei grow for pulse number up to 103 because tiny particles grow easier than larger ones. Here, collisions with atoms dominate in the growth process. The obtained results show that further increase in the number of pulses affects particle size distribution only slightly. This effect takes place if laser frequency is not too high, so that the ablated material has time to diffuse and concentration does not grow near the target. As a result, the mean radius can remain rather small (in the nanometer range, smaller than ~3 nm here).

The criterion of the catastrophic nucleation due to thermalization can be derived based on the inequality *nc* ≤ 1. This means that

$$\mathcal{N}\_c = \left[ 8\pi a^2 \sigma \;/\, 3k\_B T \ln \left( c \;/\, c\_{eq} \right) \right]^3 \sim 1. \tag{9}$$

Typically, at the beginning of the diffusional expansion stage, the condition ln(*S*)=ln(*<sup>c</sup>* / *ceq*) =8*π<sup>a</sup>* <sup>2</sup> *σ* / 3*kT* ≥1 is valid, so that this mechanism prevails in the nanoparticle formation. Finally, at the last stage, ripening or sintering of the created particles eventually inters into play if the background density is sufficiently high. For instance, this process occurs in liquids, in particular, in the absence of the surface passivation by additional chemicals.

### **5. Conclusions**

In this chapter, recent advancements in the modeling of laser ablation and spark discharges are summarized. In addition, we have compared processes leading to nanoparticle formation in laser ablation and in spark discharges. In particular, a comparison of the influence of plasma properties on nanoparticle formation has been performed.

First, mechanisms of nanoparticle generation have been investigated for femtosecond laser interactions in the presence of a background environment. The obtained calculation results have demonstrated the long time-evolution of plume species involves nucleation and growth determining the final size distribution that tends to be a limited one with the increase in laser intensity. The obtained results explain several experimental observations including both longer time dynamics of nanoparticles and size distributions. Then, conditions are formulated for catastrophic nucleation to become the main mechanism of nanoparticle formation as a result of thermalization and collisions among the species in the presence of a background environment. The produced nanoparticles can be collected, form a colloid, or can be deposited at a substrate forming nanostructures. Therefore, the presented study is of interest for many applications where both metallic nanoparticles and nanostructures are used in nanophotonics, plasmonics, medicine, biological sensing, textile industry, and other promising fields.

Furthermore, cylindrical plasma column expansion has been shown to govern primary nanoparticle formation in spark discharge, whereas hemispherical shock describes quite well this process for nanosecond laser ablation at atmospheric pressure. In addition, spark dis‐ charge leads to oscillations in plasma properties, whereas monotonous behavior is a charac‐ teristic for nanosecond laser ablation.

Despite the difference in plasma density and time evolutions calculated for both phenomena, after well-defined delays, similar critical nuclei have been shown to be formed by both techniques. This result can be attributed to the fact that whereas larger evaporation rate is typical for nanosecond laser ablation, a mixture of vapor and background gas determines the supersaturation in the case of spark.

### **Acknowledgements**

**Figure 7.** Calculated size distribution obtained for 10, 100, 1000, and 2000 pulses. Here, laser frequency is 1 kHz, gold

The obtained results (Figure 7) show that when several pulses are applied, these small nuclei

collisions with atoms dominate in the growth process. The obtained results show that further increase in the number of pulses affects particle size distribution only slightly. This effect takes place if laser frequency is not too high, so that the ablated material has time to diffuse and concentration does not grow near the target. As a result, the mean radius can remain rather

The criterion of the catastrophic nucleation due to thermalization can be derived based on the

( ) <sup>3</sup> <sup>2</sup> <sup>8</sup>

/ 3 ln ~ 1/ . = é ù

Typically, at the beginning of the diffusional expansion stage, the condition

formation. Finally, at the last stage, ripening or sintering of the created particles eventually inters into play if the background density is sufficiently high. For instance, this process occurs in liquids, in particular, in the absence of the surface passivation by additional chemicals.

In this chapter, recent advancements in the modeling of laser ablation and spark discharges are summarized. In addition, we have compared processes leading to nanoparticle formation in laser ablation and in spark discharges. In particular, a comparison of the influence of plasma

because tiny particles grow easier than larger ones. Here,

ë û *N a kT c c <sup>c</sup> <sup>B</sup> eq* (9)

*σ* / 3*kT* ≥1 is valid, so that this mechanism prevails in the nanoparticle

solution in water is considered with *a* = 1.59–10 m.

small (in the nanometer range, smaller than ~3 nm here).

properties on nanoparticle formation has been performed.

p s

grow for pulse number up to 103

10 Nanoparticles Technology

inequality *nc* ≤ 1. This means that

ln(*S*)=ln(*<sup>c</sup>* / *ceq*) =8*π<sup>a</sup>* <sup>2</sup>

**5. Conclusions**

The research leading to these results received funding from the European Union Seventh Framework Programme (FP7/2007-2013) under Grant Agreement n° 280765 (BUONAPART-E). Computer support is provided by CINES of France under the project C2015085015. Partial support from PALSE ERTIGO project (Lyon-Saint-Etienne) and PICS 6106 of CNRS, France, is also gratefully acknowledged.

### **Author details**

Andrey Voloshko and Tatiana E. Itina\*

\*Address all correspondence to: tatiana.itina@univ-st-etienne.fr

Hubert Curien Laboratory, UMR 5516 CNRS/Lyon University, Saint-Etienne, France

### **References**


### **Fundamentals of Medicinal Application of Titanium Dioxide Nanoparticles**

Kazutaka Hirakawa

**References**

12 Nanoparticles Technology

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Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/61302

### **Abstract**

Titanium dioxide (TiO2), a semiconducting material, is a well-known photocatalyst. A nanoparticle (NP) of TiO2 also demonstrates photocatalytic activity. Photo-irradiated TiO2 NPs induce the formation of various reactive species, leading to the damage of biomacromolecules. These reactive species include h+ , either free or trapped hydroxyl radicals (OH<sup>⋅</sup> ), superoxide (O2 ⋅-), hydrogen peroxide (H2O2), and singlet oxygen (1 O2), among others. TiO2 NPs photocatalyze DNA oxidation. A relatively small concentra‐ tion of TiO2 NPs frequently induces tandem base oxidation at guanine and thymine residues through H2O2 generation in the presence of a copper(II) ion. A copper–per‐ oxo complex is considered to be an important reactive species responsible for this DNA damage. In the case of a high concentration of TiO2 NPs, OH<sup>⋅</sup> contributes to DNA damage without sequence specificity. In the presence of sugars, TiO2 NPs indi‐ rectly induce DNA damage by the secondary H2O2, which is produced through an au‐ toxidation process of the product of sugar photooxidized by TiO2 NPs. Furthermore, 1 O2 is also produced by photo-irradiated TiO2 NPs. The photocatalyzed formation of 1 O2 might contribute to the oxidation of the membrane protein. These mechanisms of photocatalytic formation of the reactive species may be involved in the photocytotox‐ icity of TiO2 NPs.

**Keywords:** Titanium dioxide, Photocatalyst, Reactive oxygen species, Photomedicine, DNA damage

### **1. Introduction**

Titanium dioxide (TiO2), a semiconducting material, is a well-known photocatalyst [1-5]. Examples of previous studies about TiO2 photocatalytic reactions are listed in Table 1. A nanoparticle (NP) of TiO2 also demonstrates photocatalytic activity. Important applications of TiO2 photocatalysts are bactericidal activity [2-4, 6-12] and degradation of chemical pollutants

© 2015 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

[2-4, 13]. Related physical and chemical mechanisms have been also investigated [2-5, 14-17]. Photo-irradiated TiO2 NPs induce the formation of various reactive species, leading to the damage of biomacromolecules. These reactive species include hole (h+ ), either free or trapped hydroxyl radicals (OH<sup>⋅</sup> ), superoxide (O2 ⋅-), hydrogen peroxide (H2O2), and singlet oxygen (1 O2), among others. Hydroxyl radicals, O2 ⋅-, H2O2, and 1 O2 are the typical reactive oxygen species. TiO2 photocatalysts have been found to kill cancer cells [18-21] other than bacteria, viruses, and algae under ultraviolet-A (wavelength: 315–400 nm) illumination [2-4, 6-12]. Therefore, one of the potential applications of the TiO2 NP photocatalyst is photodynamic therapy (PDT), which is a promising treatment for cancer and some nonmalignant conditions [22-25]. In general, the mechanism of cytotoxicity by the photocatalysis of TiO2 is based on cell membrane damage via the generation of the aforementioned reactive oxygen species. Fur‐ thermore, DNA damage in human cells [26-28], mouse lymphoma cells [29], and phage [30] by the TiO2 NP photocatalyst has been reported. Direct damage of isolated DNA by TiO2 photocatalyst *in vitro* has been also studied [31, 32]. However, the DNA-damaging mechanism *in vivo* is not well-understood, because the incorporation of the TiO2 NPs in the nucleus is difficult [18]. A previous study has shown that H2O2 formation through the photocatalytic reaction of TiO2 may contribute to cellular DNA damage [2, 19]. Hydrogen peroxide, a longlived reactive oxygen species, can penetrate the nucleus membrane and induce oxidation of the nucleobase and strand breakage through enhancement by metal ions. Iron or copper ions can enhance the activity of H2O2 to produce OH<sup>⋅</sup> [33] and copper-peroxide [34-36]. Further‐ more, secondary generation of reactive oxygen species may contribute to cytotoxicity of TiO2 NPs photocatalyst [37]. Since the photocatalytic reaction will occur in a complex biological environment, an interaction between TiO2 NPs and biomaterials should participate in the generation of reactive species to induce DNA damage. For example, sugars photocatalyzed by TiO2 NPs may secondarily generate H2O2 through their further oxidation process by molecular oxygen in the presence of a metal ion [37]. In addition, the possibility of 1 O2-mediated cyto‐ toxicity by TiO2 NPs has been proposed [38]. Actually, <sup>1</sup> O2 generation by photo-irradiated TiO2 NPs was demonstrated by a near-infrared spectroscopy [39, 40]. In this chapter, recent studies about photocatalytic biomacromolecule damage by TiO2 NPs are briefly reviewed.


**Table 1.** Summary of the examples of previous studies onTiO2 photocatalyst

### **1.1. General mechanism of photocatalysis of TiO2 NP**

[2-4, 13]. Related physical and chemical mechanisms have been also investigated [2-5, 14-17]. Photo-irradiated TiO2 NPs induce the formation of various reactive species, leading to the

species. TiO2 photocatalysts have been found to kill cancer cells [18-21] other than bacteria, viruses, and algae under ultraviolet-A (wavelength: 315–400 nm) illumination [2-4, 6-12]. Therefore, one of the potential applications of the TiO2 NP photocatalyst is photodynamic therapy (PDT), which is a promising treatment for cancer and some nonmalignant conditions [22-25]. In general, the mechanism of cytotoxicity by the photocatalysis of TiO2 is based on cell membrane damage via the generation of the aforementioned reactive oxygen species. Fur‐ thermore, DNA damage in human cells [26-28], mouse lymphoma cells [29], and phage [30] by the TiO2 NP photocatalyst has been reported. Direct damage of isolated DNA by TiO2 photocatalyst *in vitro* has been also studied [31, 32]. However, the DNA-damaging mechanism *in vivo* is not well-understood, because the incorporation of the TiO2 NPs in the nucleus is difficult [18]. A previous study has shown that H2O2 formation through the photocatalytic reaction of TiO2 may contribute to cellular DNA damage [2, 19]. Hydrogen peroxide, a longlived reactive oxygen species, can penetrate the nucleus membrane and induce oxidation of the nucleobase and strand breakage through enhancement by metal ions. Iron or copper ions can enhance the activity of H2O2 to produce OH<sup>⋅</sup> [33] and copper-peroxide [34-36]. Further‐ more, secondary generation of reactive oxygen species may contribute to cytotoxicity of TiO2 NPs photocatalyst [37]. Since the photocatalytic reaction will occur in a complex biological environment, an interaction between TiO2 NPs and biomaterials should participate in the generation of reactive species to induce DNA damage. For example, sugars photocatalyzed by TiO2 NPs may secondarily generate H2O2 through their further oxidation process by molecular

⋅-, H2O2, and 1

), either free or trapped

O2-mediated cyto‐

O2 generation by photo-irradiated

O2 are the typical reactive oxygen

⋅-), hydrogen peroxide (H2O2), and singlet oxygen

damage of biomacromolecules. These reactive species include hole (h+

oxygen in the presence of a metal ion [37]. In addition, the possibility of 1

TiO2 NPs was demonstrated by a near-infrared spectroscopy [39, 40]. In this chapter, recent studies about photocatalytic biomacromolecule damage by TiO2 NPs are briefly reviewed.

toxicity by TiO2 NPs has been proposed [38]. Actually, <sup>1</sup>

**Target References** Reviews [2], [3], [4], [5]

Chemical compounds [13], [14], [15] Nucleic acids [31], [32]

Cancer cell [18], [19], [20], [21]

**Table 1.** Summary of the examples of previous studies onTiO2 photocatalyst

Mouse lymphoma cells [29] Cancer treatment of mouse [20]

Physical experiment [1], [16], [17], [39], [40]

Microorganism [6], [7], [8], [9], [10], [11], [12], [30]

), superoxide (O2

O2), among others. Hydroxyl radicals, O2

hydroxyl radicals (OH<sup>⋅</sup>

14 Nanoparticles Technology

(1

The crystal of TiO2 is a semiconductor, and the two crystalline forms, anatase and rutile, are well-known (Figure 1) [2-5]. The values of the band gap energy of these crystal forms are 3.26 and 3.06 eV for anatase and rutile, respectively. Photo-irradiation to a TiO2 crystal induces the formation of an excited electron (e- ) in the conduction band and an h+ in the valence band, leading to the redox reaction of materials adsorbing on the TiO2 surface, including water and/or molecular oxygen. The photocatalytic reactions with its surface water and oxygen cause the formation of various reactive oxygen species such as free or trapped OH<sup>⋅</sup> , O2 ⋅-, H2O2, and 1 O2 [2-5].

**Figure 1.** Band gap energy of the two crystalline forms of TiO2.

An excited electron in the conductive band reduces the oxygen molecule adsorbed on the surface of TiO2 NPs, leading to the generation of various reactive oxygen species as follows (Figure 2):

$$\mathbf{O}\_2 \mathbf{+} \mathbf{e}^\cdot \to \mathbf{O}\_2^\cdot \,\mathrm{ }^\cdot \tag{1}$$

$$\text{H}\_2\text{O}\_2^- + 2\text{H}^+ \rightarrow \text{H}\_2\text{O}\_2 + \text{O}\_2 \tag{2}$$

$$\text{CH}\_2\text{O}\_2 \rightarrow 2\text{OH}^-\tag{3}$$

The reaction (3) is mediated by ultraviolet radiation (hν, wavelength <355 nm), metal ions (Mn+) such as Fe2+, and O2 ⋅-, as follows [33]:

$$\text{H}\_2\text{O}\_2 + \text{h}\nu \to 2\text{OH}^-\tag{4}$$

$$\rm H\_2O\_2 + M^{n\*} \rightarrow OH + OH^\cdot + M^{(n+1)\*} \tag{5}$$

$$\text{H}\_2\text{O}\_2 + \text{O}\_2^- \rightarrow \text{OH}^\cdot + \text{OH}^\cdot + \text{O}\_2^\cdot \tag{6}$$

On the other hand, the formed h+ in the valence band can oxidize water to form OH<sup>⋅</sup> as follows:

$$\rm H\_2O + h^+ \to OH^\cdot + H^+ \tag{7}$$

Furthermore, OH<sup>⋅</sup> can produce H2O2 as follows:

$$\text{2OH} \rightarrow \text{H}\_2\text{O}\_2\tag{8}$$

**Figure 2.** Photocatalytic reactive oxygen formation by TiO2.

A photo-irradiated TiO2 NP can induce 1 O2 formation. The formation of 1 O2 is considered to be an important mechanism of PDT. This reaction can be explained by the following process: O2 ⋅- formed by TiO2 photocatalysis is reoxidized by the h+ of TiO2 on the particle surface to form 1 O2 as follows (Figure 3):

$$\left(\text{O}\_2\text{"} + \text{h}^+ \rightarrow {}^1\text{O}\_2\text{(}^1\text{E}\_{\text{g}}\text{"} \text{ or } {}^1\text{A}\_{\text{g}}\text{(}\text{(}\text{or }{}^3\text{O}\_2\text{)}\text{)}\right) \tag{9}$$

$$\text{\textquotedblleft O}\_2\left(^{1}\Sigma\_{\text{g}}\,^{\*}\right)\to \text{\textquotedblleft O}\_2\left(^{1}\Delta\_{\text{g}}\right) \tag{10}$$

These reactive oxygen species should contribute to the mechanism of the phototoxicity induced by TiO2 NPs.

**Figure 3.** Photocatalytic 1 O2 generation by TiO2

### **1.2. Sterilization effect by TiO2**


+ + H O + h OH + H <sup>2</sup> ® <sup>×</sup> (7)

2 2 2OH H O <sup>×</sup> ® (8)

as follows:

O2 is considered to

of TiO2 on the particle surface to

in the valence band can oxidize water to form OH<sup>⋅</sup>

Oxidation

O2 formation. The formation of 1

<sup>×</sup> ®SD (9)

O O 2g 2g S® D (10)

H2O

O2

On the other hand, the formed h+

can produce H2O2 as follows:

**Figure 2.** Photocatalytic reactive oxygen formation by TiO2.

⋅- formed by TiO2 photocatalysis is reoxidized by the h+

A photo-irradiated TiO2 NP can induce 1

O2 as follows (Figure 3):

induced by TiO2 NPs.

O2

form 1

e-

hn Reduction

O2 •−

h+

OH•

be an important mechanism of PDT. This reaction can be explained by the following process:

( )( ) - + 1 1+ <sup>1</sup> <sup>3</sup> O + h O or or O 2 2g g 2

( ) () 1 1+ 1 1

These reactive oxygen species should contribute to the mechanism of the phototoxicity

Furthermore, OH<sup>⋅</sup>

16 Nanoparticles Technology

One of the most important medicinal applications of TiO2 NPs is to kill bacteria on its surfaces. TiO2 NPs under ultraviolet radiation produce a strong oxidative effect through the formation of above-mentioned reactive oxygen species and can be used as a photocatalytic disinfectant without other chemical reagents. Fujishima and coworkers reported the bactericidal effect of TiO2 photocatalysts against *Escherichia coli* under ultraviolet-A irradiation using black light [6]. This is the first report of the application of phototoxicity of TiO2 NPs. It was speculated that H2O2 was a reactive species responsible for this phototoxic effect [7]. Relevantly, the photoca‐ talytic effect of TiO2 against methicillin-resistant *Staphylococcus aureus* (MRSA) and *Clostridium difficile* in hospitals has been reported [10]. The bactericidal effect of TiO2 NPs could be enhanced by metal doping [9]. Furthermore, visible-light-induced TiO2 photocatalysts were developed and utilized in antibacterial applications. For example, sulfur-doped TiO2 demon‐ strates the killing effect on *Escherichia coli* under white-light irradiation commonly used in hospitals [11].

### **1.3. Photodynamic therapy**

Photodynamic therapy, which is a promising and less-invasive treatment for cancer, employs a photosensitizer and visible light to produce oxidative stress in cells and ablate cancerous tumors [22-25]. Photodynamic therapy is also used for treating some nonmalignant conditions that are generally characterized by the overgrowth of unwanted or abnormal cells. In general, porphyrins are used as photosensitizers under visible-light irradiation, since the human tissue has relatively high transparency for visible light, especially red light, and visible light has hardly any side effects. In the case of visible light PDT, 1 O2 is considered an important reactive species for PDT because 1 O2 can be easily generated by visible light [41-44]. Critical targets of the generated 1 O2 include mitochondria and enzyme proteins. Moreover, DNA is also an important target biomolecule of photosensitized reactions [45-49]. Relevantly, photocatalytic 1 O2 generation by TiO2 has been reported [38-40].

TiO2, a nontoxic material, is chemically stable, and demonstrates a phototoxic effect. Therefore, an application of TiO2 for PDT has been investigated [2]. The cytotoxicity of an illuminated TiO2 film electrode for HeLa cells [18,19] and T-24 human bladder cancer cells [21] has been reported. Animal experiments also demonstrated the antitumor effect of TiO2 NPs [20]. This report showed an antineoplastic effect on skin cancer in mouse models.

### **2. Photocatalytic DNA damage by TiO2 NPs**

Cellular DNA damage photocatalyzed by TiO2 NPs was demonstrated by the experiment using cancer cells [18,19,21]. TiO2 NPs can be taken into the cancer cell [27]; however, incorporation into the cell nucleus is difficult [18]. Therefore, it is speculated that the indirect mechanism contributes to DNA damage induced by photo-irradiated TiO2 NPs. Hence, model experiments using isolated DNA were performed [31, 32]. In this section, an example of photocatalytic DNA damage by TiO2 NPs was introduced.

### **2.1. Isolated DNA damage photocatalyzed by TiO2 NPs and its sequence specificity**

Photo-irradiated TiO2 NPs catalyze DNA damage in the presence of copper(II) ion [31]. Relevantly, copper-aided photosterilization of microbial cells on TiO2 was reported [8]. DNA damage by anatase NPs is more severe than that by rutile NPs. The DNA damage is enhanced by piperidine treatment, because photo-irradiated TiO2 NPs cause not only DNA strand breakage but also base oxidation. In general, hot piperidine cleaves DNA strand at modified base. Photo-irradiated TiO2 NPs induce the formation of piperidine-labile products at the bolded site of 5'-**T**G, 5'-T**G**, and 5'-**T**C (Figure 4). Furthermore, TiO2 NPs photocatalyze DNA strand cleavage at the bolded guanines of 5'-T**G** and 5'-T**C** in a DNA fragment treated with *E coli* formamidopyrimidine-DNA glycosylase (Fpg protein), which can catalyze the excision of piperidine-resistant 8-oxo-7,8-dihydro-2'deoxyguanine (8-oxo-G) [50,51]. The formation of 8 oxo-G was confirmed by an analysis with a high-performance liquid chromatography (Figure 5). In addition, Fpg protein can cleave the oxidized cytosine, such as 5-hydroxy cytosine [52]. These results suggest that photo-irradiated TiO2 NPs induce 8-oxo-G formation adjacent to piperidine-labile thymine lesions. Such double-base lesions should be generated from one radical hit that leads through a secondary reaction to a tandem base damage at pyrimidine and adjacent residues [53-56]. Actually, it has been reported that H2O2 induces tandem mutations in human cells via vicinal or cross-linked base modification in the presence of copper(II) ion [57]. Since repairing of cluster DNA damage in living cells is difficult [58], such clustered base damage, including double-base lesions, appears to play an important role in the phototoxicity of TiO2 NPs.

### **2.2. Mechanism of DNA damage photocatalyzed by TiO2 NPs**

Catalase, a well-known scavenger of H2O2, and bathocuproines, a copper(I) ion chelator, inhibit DNA damage photocatalyzed by TiO2 NPs, whereas, typical OH<sup>⋅</sup> scavenger cannot inhibit the DNA damage. These results suggest that H2O2 and copper(I) ion participate in DNA damage

TiO2, a nontoxic material, is chemically stable, and demonstrates a phototoxic effect. Therefore, an application of TiO2 for PDT has been investigated [2]. The cytotoxicity of an illuminated TiO2 film electrode for HeLa cells [18,19] and T-24 human bladder cancer cells [21] has been reported. Animal experiments also demonstrated the antitumor effect of TiO2 NPs [20]. This

Cellular DNA damage photocatalyzed by TiO2 NPs was demonstrated by the experiment using cancer cells [18,19,21]. TiO2 NPs can be taken into the cancer cell [27]; however, incorporation into the cell nucleus is difficult [18]. Therefore, it is speculated that the indirect mechanism contributes to DNA damage induced by photo-irradiated TiO2 NPs. Hence, model experiments using isolated DNA were performed [31, 32]. In this section, an example of photocatalytic DNA

**2.1. Isolated DNA damage photocatalyzed by TiO2 NPs and its sequence specificity**

Photo-irradiated TiO2 NPs catalyze DNA damage in the presence of copper(II) ion [31]. Relevantly, copper-aided photosterilization of microbial cells on TiO2 was reported [8]. DNA damage by anatase NPs is more severe than that by rutile NPs. The DNA damage is enhanced by piperidine treatment, because photo-irradiated TiO2 NPs cause not only DNA strand breakage but also base oxidation. In general, hot piperidine cleaves DNA strand at modified base. Photo-irradiated TiO2 NPs induce the formation of piperidine-labile products at the bolded site of 5'-**T**G, 5'-T**G**, and 5'-**T**C (Figure 4). Furthermore, TiO2 NPs photocatalyze DNA strand cleavage at the bolded guanines of 5'-T**G** and 5'-T**C** in a DNA fragment treated with *E coli* formamidopyrimidine-DNA glycosylase (Fpg protein), which can catalyze the excision of piperidine-resistant 8-oxo-7,8-dihydro-2'deoxyguanine (8-oxo-G) [50,51]. The formation of 8 oxo-G was confirmed by an analysis with a high-performance liquid chromatography (Figure 5). In addition, Fpg protein can cleave the oxidized cytosine, such as 5-hydroxy cytosine [52]. These results suggest that photo-irradiated TiO2 NPs induce 8-oxo-G formation adjacent to piperidine-labile thymine lesions. Such double-base lesions should be generated from one radical hit that leads through a secondary reaction to a tandem base damage at pyrimidine and adjacent residues [53-56]. Actually, it has been reported that H2O2 induces tandem mutations in human cells via vicinal or cross-linked base modification in the presence of copper(II) ion [57]. Since repairing of cluster DNA damage in living cells is difficult [58], such clustered base damage, including double-base lesions, appears to play an important role in

Catalase, a well-known scavenger of H2O2, and bathocuproines, a copper(I) ion chelator, inhibit

DNA damage. These results suggest that H2O2 and copper(I) ion participate in DNA damage

scavenger cannot inhibit the

report showed an antineoplastic effect on skin cancer in mouse models.

**2. Photocatalytic DNA damage by TiO2 NPs**

damage by TiO2 NPs was introduced.

18 Nanoparticles Technology

the phototoxicity of TiO2 NPs.

**2.2. Mechanism of DNA damage photocatalyzed by TiO2 NPs**

DNA damage photocatalyzed by TiO2 NPs, whereas, typical OH<sup>⋅</sup>

**Figure 4.** Sequence specificity of DNA damage photocatalyzed by anatase TiO2 NPs. The 32P-end-labeled 211 base pair DNA fragment (*p53* tumor suppressor gene) and 8 μg mL-1 anatase was irradiated with ultraviolet light (365 nm, 10 J cm-2) with 20 μM copper(II) ion in a 10 mM sodium phosphate buffer (pH 7.8). After the photocatalytic reaction, the DNA fragments were treated with hot piperidine or Fpg and analyzed by an electrophoresis.

**Figure 5.** Formation of 8-oxo-G by the photocatalytic reaction of anatase or rutile NPs. Calf thymus DNA was treated by the photocatalytic reaction of anatase or rutile NPs (365 nm, 10 J cm-2) with 20 μM copper(II) ion in a 10 mM sodium phosphate buffer (pH 7.8). After the photocatalytic reaction, the samples were analyzed with a high-performance liq‐ uid chromatography.

by photo-irradiated TiO2 NPs. It has been reported that OH<sup>⋅</sup> is not the main reactive species involved in DNA damage by H2O2 and copper(I) ions [34-36, 59]. DNA-associated copper(I) ions may generate other oxidants, including a copper–peroxo intermediate, such as Cu(I)- OOH, which is generated from the reaction of H2O2 and copper(I) ions [34-36, 59]. Indeed, methional, which can scavenge Cu(I)-OOH [36, 59], shows inhibitory effect on DNA damage photocatalyzed by TiO2 NPs. The generation of these reactive species may be responsible for the formation of piperidine-labile products and 8-oxo-G.

On the other hand, a high concentration of anatase NPs can catalyze DNA photodamage without copper(II) ions. Typical OH<sup>⋅</sup> scavengers, ethanol and sugars, effectively inhibit the DNA photodamage by a high concentration of anatase NPs. The DNA damage induced by photo-irradiated anatase NPs without copper(II) ions is observed at every nucleobases without site specificity. Such DNA damage without sequence-specificity is the typical pattern of OH<sup>⋅</sup> mediated DNA damage [34].

A proposed mechanism of DNA damage photocatalyzed by TiO2 NPs is shown in Figure 6. The crystalline forms of TiO2, anatase and rutile, are semiconductors with band gap energies of 3.26 and 3.06 eV, which correspond to the following wavelengths of light: 385 and 400 nm, respectively. When a TiO2 semiconductor NPs absorbs photon with energy greater than their band gap, electrons in the valence band are excited to the conduction band, creating electronh+ pairs and causing various chemical reactions [2-5]. The electron acts as a reductant, whereas the h+ is a powerful oxidant. In aqueous environments, oxygen molecule can be reduced by the electron into O2 ⋅-, and water molecule can be oxidized by the h+ into OH<sup>⋅</sup> . In general, formed O2 ⋅- can be dismutated into H2O2 by proton. The oxygen reduction may precede the reduction of copper(II) ions under aerobic condition, since the concentration of dissolved oxygen is higher (~250 μM) than that of the copper(II) ion used in this study (20 μM). The copper(II) reduction may be mediated by O2 ⋅-. Hydrogen peroxide reacts with copper(I) ions to generate other oxidants, including a copper–peroxo intermediate, resulting in the oxidation of DNA bases. Copper ions, which are essential components of chromatin [60,61], are found to bind DNA with high affinity [62,63]. Therefore, copper ions may play an important role in reactive oxygen generation *in vivo*, although mammals have evolved means of minimizing the levels of free copper ions and most copper ions bind to protein caries and transporters [64]. Hydroxyl radicals formed by the reaction of water with an h+ in the valence band of TiO2 NPs also slightly participate in DNA damage photocatalyzed by anatase NPs. Because OH<sup>⋅</sup> is a strong oxidative agent, OH<sup>⋅</sup> can damage every nucleobase [34]. The present results suggest that H2O2 mainly participate in the phototoxicity of TiO2 NPs and the contribution of OH<sup>⋅</sup> is relatively small. Fujishima *et al.* also reported the involvement of H2O2 generated from O2 ⋅- in the cytotoxicity of illuminated TiO2 NPs [2-4, 8-13].

TiO2 NPs might be a potential agent for PDT [22-25]. TiO2 NPs can be incorporated into cancer cells and demonstrate cytotoxicity under photo-irradiation [2-4, 26-28]. Photocatalytic reaction by TiO2 NPs induces a number of functional changes in cell including altered permeability of cellular membranes to potassium and calcium ions, release of RNA and proteins and cytotox‐ icity [2,18-21]. It has been reported that DNA can be a target biomolecule of the photocatalytic reaction of TiO2 NPs [26-30]. Although incorporation of TiO2 NPs into cell nucleus is difficult [18], the generated H2O2 by a photocatalytic reaction of TiO2 NPs can be easily diffused and incorporated in a cell nucleus, leading to DNA photodamage with metal ions. Relevantly, several studies demonstrated that DNA is a potential target of PDT [47,65,66]. Therefore, the

**Figure 6.** Proposed mechanism of DNA damage photocatalyzed by TiO2 NPs.

methional, which can scavenge Cu(I)-OOH [36, 59], shows inhibitory effect on DNA damage photocatalyzed by TiO2 NPs. The generation of these reactive species may be responsible for

On the other hand, a high concentration of anatase NPs can catalyze DNA photodamage

DNA photodamage by a high concentration of anatase NPs. The DNA damage induced by photo-irradiated anatase NPs without copper(II) ions is observed at every nucleobases without site specificity. Such DNA damage without sequence-specificity is the typical pattern of OH<sup>⋅</sup>

A proposed mechanism of DNA damage photocatalyzed by TiO2 NPs is shown in Figure 6. The crystalline forms of TiO2, anatase and rutile, are semiconductors with band gap energies of 3.26 and 3.06 eV, which correspond to the following wavelengths of light: 385 and 400 nm, respectively. When a TiO2 semiconductor NPs absorbs photon with energy greater than their band gap, electrons in the valence band are excited to the conduction band, creating electron-

pairs and causing various chemical reactions [2-5]. The electron acts as a reductant, whereas

⋅- can be dismutated into H2O2 by proton. The oxygen reduction may precede the reduction of copper(II) ions under aerobic condition, since the concentration of dissolved oxygen is higher (~250 μM) than that of the copper(II) ion used in this study (20 μM). The copper(II)

other oxidants, including a copper–peroxo intermediate, resulting in the oxidation of DNA bases. Copper ions, which are essential components of chromatin [60,61], are found to bind DNA with high affinity [62,63]. Therefore, copper ions may play an important role in reactive oxygen generation *in vivo*, although mammals have evolved means of minimizing the levels of free copper ions and most copper ions bind to protein caries and transporters [64]. Hydroxyl

participate in DNA damage photocatalyzed by anatase NPs. Because OH<sup>⋅</sup> is a strong oxidative

TiO2 NPs might be a potential agent for PDT [22-25]. TiO2 NPs can be incorporated into cancer cells and demonstrate cytotoxicity under photo-irradiation [2-4, 26-28]. Photocatalytic reaction by TiO2 NPs induces a number of functional changes in cell including altered permeability of cellular membranes to potassium and calcium ions, release of RNA and proteins and cytotox‐ icity [2,18-21]. It has been reported that DNA can be a target biomolecule of the photocatalytic reaction of TiO2 NPs [26-30]. Although incorporation of TiO2 NPs into cell nucleus is difficult [18], the generated H2O2 by a photocatalytic reaction of TiO2 NPs can be easily diffused and incorporated in a cell nucleus, leading to DNA photodamage with metal ions. Relevantly, several studies demonstrated that DNA is a potential target of PDT [47,65,66]. Therefore, the

participate in the phototoxicity of TiO2 NPs and the contribution of OH<sup>⋅</sup>

Fujishima *et al.* also reported the involvement of H2O2 generated from O2

can damage every nucleobase [34]. The present results suggest that H2O2 mainly

⋅-, and water molecule can be oxidized by the h+

is a powerful oxidant. In aqueous environments, oxygen molecule can be reduced by

scavengers, ethanol and sugars, effectively inhibit the

into OH<sup>⋅</sup>

in the valence band of TiO2 NPs also slightly

⋅-. Hydrogen peroxide reacts with copper(I) ions to generate

. In general, formed

is relatively small.

⋅- in the cytotoxicity


the formation of piperidine-labile products and 8-oxo-G.

without copper(II) ions. Typical OH<sup>⋅</sup>

mediated DNA damage [34].

20 Nanoparticles Technology

h+

O2

the h+

agent, OH<sup>⋅</sup>

the electron into O2

reduction may be mediated by O2

of illuminated TiO2 NPs [2-4, 8-13].

radicals formed by the reaction of water with an h+

metal-mediated DNA damage through the photocatalysis of TiO2 NPs may participate in cytotoxicity by photo-irradiated TiO2 NPs.

### **3. Secondary production of reactive oxygen species from photocatalyzed materials by TiO2 NPs**

As mentioned above, DNA damage in human cells by TiO2 NPs has also been reported [26-28]. The direct DNA damage by TiO2 NPs photocatalyst *in vitro* has been also studied [31, 32]. However, the DNA-damaging mechanism *in vivo* is not well-understood because the incor‐ poration of the TiO2 NPs in the cell nucleus is difficult [18]. Since the TiO2 photocatalytic reaction occurs in a complex biological environment, an interaction between TiO2 NPs and biomaterials may participate in the generation of reactive species to induce DNA damage. Hence, the effect of sugars, which are ubiquitous biomaterials, on DNA damage photocata‐ lyzed by TiO2 NPs was examined [37].

In the case of anatase, a high concentration of TiO2 NPs can damage DNA at every nucleobase by OH<sup>⋅</sup> generation in the absence of copper(II) ions. Typical free OH<sup>⋅</sup> scavengers inhibited this copper(II)-independent DNA damage. These results indicate that free OH<sup>⋅</sup> partly contributes to DNA damage photocatalyzed by TiO2. On the other hand, scavengers of OH<sup>⋅</sup> , such as a sugar (mannitol), ethanol, and formate, enhanced the copper(II)-dependent DNA damage [31]. These scavengers themselves did not induce DNA damage. Since OH<sup>⋅</sup> can oxidize most biomaterials, the oxidized products of biomaterials by the TiO2 photocatalyst may damage DNA via the generation of secondary reactive oxygen species. The addition of sugars, glucose and galactose, which are ubiquitous biomolecules, enhanced the DNA damage photocatalyzed by TiO2 NPs. Enhancement of DNA damage by sugars has seldom been reported, and these sugars themselves could not induce DNA damage. Therefore, the products of the photocata‐ lytic reaction of these sugars by TiO2 NPs is responsible for the copper(II)-dependent damage to DNA. Indeed, the glucose and galactose oxidized by the TiO2 photocatalytic reaction caused DNA damage in the presence of copper(II) ion [37]. The inhibitory effect of various scavengers for DNA damage by the photo-oxidized products of sugars by TiO2 was examined. Catalase inhibited DNA damage by the photocatalyzed glucose, indicating the involvement of H2O2. Bathocuproine, which is a chelator of copper(I) ion, also inhibited DNA damage by the photocatalyzed glucose, suggesting the involvement of copper(I) ion. The free OH<sup>⋅</sup> scavengers had no or little inhibitory effect on DNA damage. The inhibitory effect of superoxide dismutase (SOD) was weak, suggesting that O2 ⋅- itself is not the main reactive species for DNA damage. Similar results were observed in the case of galactose. Fluorometry using folic acid [67] demonstrated the formation of H2O2 from the photocatalyzed sugars (Figure 7). The amount of H2O2 generation was comparable with that of other H2O2-mediated DNA-damaging drugs [68]. H2O2 generation was not observed in the absence of copper(II) ions. These results showed that the oxidized products of sugars generate H2O2 during the reaction with copper(II) ions, resulting in secondary DNA damage.

**Figure 7.** Hydrogen peroxide generation from photo-oxidized glucose and galactose by TiO2 NPs. The buffer solution with 10 mM sugars was previously irradiated (365 nm, 6 J cm-2) with 100 μg mL-1 anatase NPs. The TiO2 NPs were removed by centrifugation, and the solution containing the oxidized sugars was used. One mL of solution containing the treated sugars and 10 μM of folic acid was incubated (60 min, 37 °C) in the presence of 20 μM copper(II) chloride, and the fluorescence intensity was measured (excitation: 360 nm, detection: 450 nm). The concentration of the generat‐ ed H2O2 was determined by the calibration curve method.

These sugars act as an electron donor for the photocatalytic reaction [15,37]. Partially oxidized sugars, such as aldehyde compounds, are possibly produced through this photocatalytic oxidation. The mechanism of DNA damage by the photocatalyzed product of sugars is proposed in Figure 8. Aldehydes can generate H2O2 via its further oxidation [69], though these sugars themselves are stable compounds. Many studies have reported DNA damage by H2O2 and copper(II) ions [34-36, 70]. Various chemical compounds, including aldehydes, easily produce O2 ⋅- through their autoxidation process. The autoxidation is markedly enhanced by copper(II) ion, which is an essential component of chromatin [60, 61]. The formed O2 ⋅- is rapidly dismutated into H2O2. Although the generated H2O2 itself cannot damage DNA, H2O2 reduces copper(II) into copper(I), leading to the activation of H2O2 through the formation of reactive species, such as Cu(I)-OOH [34-36, 59]. Indeed, methional, a scavenger of Cu(I)-OOH, inhibited the DNA damage. This reactive species cannot be scavenged by the free OH<sup>⋅</sup> scavengers; however, it can effectively oxidize the nucleobases [34-36, 59].

**Figure 8.** Proposed mechanism of secondary DNA damage by photocatalyzed sugars.

DNA damage in the presence of copper(II) ion [37]. The inhibitory effect of various scavengers for DNA damage by the photo-oxidized products of sugars by TiO2 was examined. Catalase inhibited DNA damage by the photocatalyzed glucose, indicating the involvement of H2O2. Bathocuproine, which is a chelator of copper(I) ion, also inhibited DNA damage by the

had no or little inhibitory effect on DNA damage. The inhibitory effect of superoxide dismutase

Similar results were observed in the case of galactose. Fluorometry using folic acid [67] demonstrated the formation of H2O2 from the photocatalyzed sugars (Figure 7). The amount of H2O2 generation was comparable with that of other H2O2-mediated DNA-damaging drugs [68]. H2O2 generation was not observed in the absence of copper(II) ions. These results showed that the oxidized products of sugars generate H2O2 during the reaction with copper(II) ions,

> Oxidized glucose Oxidized galactose

[Photocatalyzed sugars] / mM Control 0.1 0.2 0.5 1.0

**Figure 7.** Hydrogen peroxide generation from photo-oxidized glucose and galactose by TiO2 NPs. The buffer solution with 10 mM sugars was previously irradiated (365 nm, 6 J cm-2) with 100 μg mL-1 anatase NPs. The TiO2 NPs were removed by centrifugation, and the solution containing the oxidized sugars was used. One mL of solution containing the treated sugars and 10 μM of folic acid was incubated (60 min, 37 °C) in the presence of 20 μM copper(II) chloride, and the fluorescence intensity was measured (excitation: 360 nm, detection: 450 nm). The concentration of the generat‐

These sugars act as an electron donor for the photocatalytic reaction [15,37]. Partially oxidized sugars, such as aldehyde compounds, are possibly produced through this photocatalytic oxidation. The mechanism of DNA damage by the photocatalyzed product of sugars is proposed in Figure 8. Aldehydes can generate H2O2 via its further oxidation [69], though these sugars themselves are stable compounds. Many studies have reported DNA damage by H2O2 and copper(II) ions [34-36, 70]. Various chemical compounds, including aldehydes, easily

dismutated into H2O2. Although the generated H2O2 itself cannot damage DNA, H2O2 reduces copper(II) into copper(I), leading to the activation of H2O2 through the formation of reactive

copper(II) ion, which is an essential component of chromatin [60, 61]. The formed O2

⋅- through their autoxidation process. The autoxidation is markedly enhanced by

⋅- itself is not the main reactive species for DNA damage.

scavengers

⋅- is rapidly

photocatalyzed glucose, suggesting the involvement of copper(I) ion. The free OH<sup>⋅</sup>

(SOD) was weak, suggesting that O2

22 Nanoparticles Technology

resulting in secondary DNA damage.

[H

0

ed H2O2 was determined by the calibration curve method.

produce O2

50

100

150

200

250

300

O2 2] /

m

M

Although TiO2 is not likely to be incorporated in a cell nucleus [18], H2O2 generated via a photocatalytic reaction can be easily diffused and incorporated in a cell nucleus. This DNAdamaging mechanism via H2O2 generation may participate in the phototoxicity of TiO2. *In vivo*, the cell membrane is an important reaction field for the TiO2 photocatalyst because TiO2 NPs show affinity with a cell membrane [18]. Further, a part of the TiO2 NPs can become incorporated into the cell. Sugars on the cell membrane and cytoplasm may be oxidized by the TiO2 photocatalytic reaction. The generated h+ and OH<sup>⋅</sup> can oxidize these sugars, leading to the formation of secondary H2O2 from their photo-oxidized products.

In summary, sugars enhance the DNA damage photocatalyzed by TiO2 NPs. This enhancement of DNA damage is due to the secondary generation of a reactive oxygen species, H2O2, which can diffuse in the cell and damage cellular DNA. These findings suggest that the secondary H2O2 generation contributes to the phototoxicity of TiO2 more than the direct formation of reactive oxygen species does.

### **4. Singlet oxygen formation through photocatalytic reaction of TiO2 NPs**

A contribution of 1 O2 in the TiO2 photocatalytic reaction was reported [38]. Singlet oxygen generation by TiO2 photocatalysis has been demonstrated by the emission measurement of 1 O2, which is assigned to the transition from 1 O2( 1 Δg) to 3 O2( 3 ∑g) [39, 40]. Because 1 O2 is considered to be an important reactive species in PDT process [22-25], the clarification of the contribution of 1 O2 generated by TiO2 photocatalysis is closely related to a design of photoca‐ talyst for medicinal application. Thus, 1 O2 generation in the TiO2 photocatalysis and its importance on biomolecular damage was examined [40].

The typical emission of 1 O2 at around 1270 nm was observed during irradiation of TiO2 NPs. Relatively strong emission of 1 O2 was observed in nonpolar organic solvents such as dichloro‐ methane. The quantum yield (ΦΔ) of 1 O2 generation by TiO2 photocatalysis in ethanol was estimated from the comparison of 1 O2 emission intensities by TiO2 NPs and methylene blue (ΦΔ= 0.52) [71] and the absorbance of the TiO2 NP dispersions. Because the scattering by suspended TiO2 NPs makes the calculation of absorbed light intensity complex, the precise estimation of the ΦΔ is difficult. Thus, the ΦΔ was estimated using the apparent absorbance of TiO2 NPs. The calculated value indicates the lowest limit of the ΦΔ by TiO2 photocatalysis in ethanol. The reported lifetime of 1 O2 generated via TiO2 photocatalytic reaction is 5 μs [39]. This value is shorter than that by the photosensitized reaction of methylene blue (12 μs) [72]. Since the emission intensity of 1 O2 is proportional to its lifetime, the ΦΔ was corrected by the lifetime of 1 O2. The estimated value of ΦΔ by both types of TiO2, anatase and rutile, was about 0.02 in ethanol. This value of ΦΔ is enough large to induce oxidative damage to biomolecules. The 1 O2 emission in D2O was completely quenched by the addition of SOD, which is the enzyme to dismutate O2 ⋅- into H2O2. These results can be explained by the fact that 1 O2 is formed by the reoxidation of O2 ⋅-, generated from the photoreduction of oxygen molecules by TiO2 NPs (Figure 3). The intensity of 1 O2 emission observed in the case of rutile was significantly larger than that by anatase in D2O. The difference of the 1 O2 generation by these two types of TiO2 crystalline forms can be reasonably explained by that in aqueous solution. H2O2 generation proceeds in the photocatalysis of anatase rather than O2 ⋅- generation, whereas O2 ⋅- is the main product from oxygen photoreduction mediated by rutile [17]. These results support the mechanism of 1 O2 generation via O2 ⋅- by TiO2 photocatalysis.

The emission spectrum of 1 O2 by TiO2 (in both, anatase and rutile type cases) slightly blueshifted (~4 nm) compared with that by methylene blue. These results suggest that the sur‐ roundings of the 1 O2 generated on the TiO2 surface are different from that by methylene blue in solution. In the case of the photosensitization of methylene blue, the generated 1O2 deacti‐ vates in the homogeneous media of solvents. A possible explanation of the blue-shift is that most of the 1O2 generated by TiO2 NPs deactivates on the TiO2 surface.

The intensity of 1 O2 emission by TiO2 photocatalysis in liposome was significantly larger than that in an aqueous solution in both, anatase and rutile type cases. The enhancement of the 1 O2 emission can be explained by the elongation of the lifetime of 1 O2 or the acceleration of the photocatalytic reaction. This result shows that phospholipids membrane is an important environment of the phototoxic reaction mediated by 1 O2 in the photocatalytic reactions of TiO2 NPs. Indeed, high affinity of TiO2 NPs with a cell membrane was reported [18]. Conse‐ quently, an environmental effect of a cell membrane is important for the photocatalytic reaction of TiO2 NPs. Since amino acid residues in proteins can be oxidized by 1 O2 [42], a membrane protein should be the target biomolecule in cell membrane. Indeed, 1 O2 emission was quenched by the addition of bovine serum albumin, a typical water soluble protein, suggesting scav‐ enging of the 1 O2 generated by TiO2 photocatalysis through oxidation of protein.

*In vivo*, nicotinamide adenine dinucleotide (NADH) is one of the most important target biomolecule oxidized by 1 O2 [73, 74]. NADH demonstrates the typical absorption peak at around 340 nm in an ultraviolet absorption spectral measurement, and this absorption band is diminished by the oxidation. It has been reported that TiO2 NPs hardly induce the oxidation of NADH in aqueous solution during ultraviolet irradiation. Since NADH hardly adsorbed on a surface of TiO2 NPs, the 1 O2 could not effectively oxidize NADH in solution. As mentioned above, it has been reported that photo-irradiated TiO2 NPs can induce DNA damage mainly through H2O2 and OH<sup>⋅</sup> , and the 1 O2-mediated DNA damage could not be observed [31]. These reports concluded that the photocatalytic 1 O2 generation on the surface of TiO2 NPs is not important in the damaging mechanism of the biomolecules such as DNA and NADH, of which the affinity with TiO2 surface is small.

In conclusion, photo-irradiated TiO2 NPs can produce 1 O2 through reoxidation of O2 ⋅-, which is formed by photocatalytic reduction of oxygen molecule on the surface of TiO2 NPs. Since most of the 1 O2 deactivated on TiO2 surface, the 1 O2 on TiO2 surface cannot induce the oxidation of DNA and NADH. However, the 1 O2 generation by TiO2 photocatalysis could be enhanced in the microenvironment of phospholipids membrane. These findings suggest that 1 O2 may contribute to phototoxicity of TiO2 NPs through oxidation of membrane protein.

### **5. Conclusions**

contribution of 1

24 Nanoparticles Technology

The typical emission of 1

Relatively strong emission of 1

talyst for medicinal application. Thus, 1

methane. The quantum yield (ΦΔ) of 1

estimated from the comparison of 1

ethanol. The reported lifetime of 1

Since the emission intensity of 1

lifetime of 1

to dismutate O2

mechanism of 1

roundings of the 1

The intensity of 1

enging of the 1

reoxidation of O2

(Figure 3). The intensity of 1

The emission spectrum of 1

than that by anatase in D2O. The difference of the 1

O2 generation via O2

proceeds in the photocatalysis of anatase rather than O2

The 1

importance on biomolecular damage was examined [40].

O2 generated by TiO2 photocatalysis is closely related to a design of photoca‐

(ΦΔ= 0.52) [71] and the absorbance of the TiO2 NP dispersions. Because the scattering by suspended TiO2 NPs makes the calculation of absorbed light intensity complex, the precise estimation of the ΦΔ is difficult. Thus, the ΦΔ was estimated using the apparent absorbance of TiO2 NPs. The calculated value indicates the lowest limit of the ΦΔ by TiO2 photocatalysis in

This value is shorter than that by the photosensitized reaction of methylene blue (12 μs) [72].

0.02 in ethanol. This value of ΦΔ is enough large to induce oxidative damage to biomolecules.

crystalline forms can be reasonably explained by that in aqueous solution. H2O2 generation

product from oxygen photoreduction mediated by rutile [17]. These results support the

shifted (~4 nm) compared with that by methylene blue. These results suggest that the sur‐

in solution. In the case of the photosensitization of methylene blue, the generated 1O2 deacti‐ vates in the homogeneous media of solvents. A possible explanation of the blue-shift is that

that in an aqueous solution in both, anatase and rutile type cases. The enhancement of the 1

photocatalytic reaction. This result shows that phospholipids membrane is an important

TiO2 NPs. Indeed, high affinity of TiO2 NPs with a cell membrane was reported [18]. Conse‐ quently, an environmental effect of a cell membrane is important for the photocatalytic reaction

by the addition of bovine serum albumin, a typical water soluble protein, suggesting scav‐

O2 generated by TiO2 photocatalysis through oxidation of protein.

most of the 1O2 generated by TiO2 NPs deactivates on the TiO2 surface.

of TiO2 NPs. Since amino acid residues in proteins can be oxidized by 1

protein should be the target biomolecule in cell membrane. Indeed, 1

emission can be explained by the elongation of the lifetime of 1

environment of the phototoxic reaction mediated by 1

⋅- by TiO2 photocatalysis.

⋅- into H2O2. These results can be explained by the fact that 1

O2 emission in D2O was completely quenched by the addition of SOD, which is the enzyme

O2. The estimated value of ΦΔ by both types of TiO2, anatase and rutile, was about

⋅-, generated from the photoreduction of oxygen molecules by TiO2 NPs

O2 generated on the TiO2 surface are different from that by methylene blue

O2 emission by TiO2 photocatalysis in liposome was significantly larger than

O2 emission observed in the case of rutile was significantly larger

O2 by TiO2 (in both, anatase and rutile type cases) slightly blue-

O2 generation by these two types of TiO2

⋅- generation, whereas O2

O2 at around 1270 nm was observed during irradiation of TiO2 NPs.

O2 was observed in nonpolar organic solvents such as dichloro‐

O2 generation in the TiO2 photocatalysis and its

O2 generation by TiO2 photocatalysis in ethanol was

O2 emission intensities by TiO2 NPs and methylene blue

O2 generated via TiO2 photocatalytic reaction is 5 μs [39].

O2 is formed by the

⋅- is the main

O2

O2 or the acceleration of the

O2 [42], a membrane

O2 emission was quenched

O2 in the photocatalytic reactions of

O2 is proportional to its lifetime, the ΦΔ was corrected by the

TiO2 NPs photocatalyze DNA oxidation. A relatively small concentration of TiO2 NPs fre‐ quently induces tandem base oxidation at guanine and thymine residues through H2O2 generation in the presence of a copper(II) ion. A copper–peroxo complex is considered to be an important reactive species responsible for this DNA damage. In addition, cytosine residues are also photooxidized by TiO2 NPs. In the case of a high concentration of TiO2 NPs, OH<sup>⋅</sup> contributes to DNA damage without sequence specificity. In the presence of sugars, TiO2 NPs indirectly induce DNA damage by the secondary H2O2, which is produced through an autoxidation process of the photo-oxidized products of sugars by TiO2 NPs. Furthermore, 1 O2 is also produced by photo-irradiated TiO2 NPs. The 1 O2 generation is explained by the reoxidation of O2 ⋅-, which is produced by photocatalytic reduction of the oxygen molecule adsorbed on the surface of TiO2 NPs. The photocatalyzed formation of 1 O2 might contribute to the oxidation of the membrane protein. These mechanisms of photocatalytic reactive oxygen formation should be involved in the photocytotoxicity of TiO2 NPs. Because TiO2 is a chemi‐ cally stable and nontoxic material, the bactericidal activity and cytotoxicity against cancer cells will play more important roles in the field of medical applications of nanomaterials.

### **Acknowledgements**

The author wishes to thank Professor Shosuke Kawanishi (Suzuka University of Medical Science) for his helpful discussion about DNA damage. The reported works were supported by a Grant-in-Aid for Scientific Research on Priority Areas (417) from the Ministry of Educa‐ tion, Culture, Sports, Science, and Technology (MEXT) of the Japanese Government.

### **Author details**

Kazutaka Hirakawa1,2

Address all correspondence to: hirakawa.kazutaka@shizuoka.ac.jp

1 Applied Chemistry and Biochemical Engineering Course, Department of Engineering, Graduate School of Integrated Science and Technology, Shizuoka University, Johoku, Nakaku, Hamamatsu, Shizuoka, Japan

2 Department of Optoelectronics and Nanostructure Science, Graduate School of Science and Technology, Shizuoka University, Johoku, Naka-ku, Hamamatsu, Shizuoka, Japan

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2 Department of Optoelectronics and Nanostructure Science, Graduate School of Science and Technology, Shizuoka University, Johoku, Naka-ku, Hamamatsu, Shizuoka, Japan

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