**Control of Fluorescence and Photosensitized Singlet Oxygen-Generating Activities of Porphyrins by DNA: Fundamentals for "Theranostics"**

Kazutaka Hirakawa

[19] Vogelaar B, Eijsbouts S, Bergwerff J, Heiszwolf J. Hydroprocessing catalyst deactivation in commercial practice. Catalysis Today. 2010;**154**:256-263. DOI: 10.1016/j.cattod.2010.03.039

[20] Furimsky E, Massoth F. Deactivation of hydroprocessing catalysts. Catalysis Today.

[21] Abdrabo A, Husein M. Method for converting demetallization products into dispersed metal oxide nanoparticles in heavy oil. Energy and Fuels. 2012;**26**:810-815.

[22] Shang H, Liu Y, Shi J, Shi Q, Zhang W. Microwave-assisted nickel and vanadium removal from crude oil. Fuel Processing Technology. 2016;**142**:250-257. DOI: 10.1016/j.fuproc.2015.09.033

[23] Shiraishi Y, Hirai T, Komasawa I. Novel demetalation process for vanadyl and nickel porphyrins from petroleum residue by photochemical reaction and liquid - liquid extraction. Industrial & Engineering Chemistry Research 2000;**39**:1345-1355. DOI: 10.1021/ie990809o

[24] Milordov D, Usmanova G, Yakubova S, Yakubov M, Romanov G. Comparative analysis of extractive methods of porphyrin separation from heavy oil asphaltenes. Chemistry and Technology of Fuels and Oils. 2013;**3**:232-238. DOI: 10.1007/s10553-013-0435-7 [25] Yakubov M, Milordov D, Yakubova S, Borisov D, Gryaznov G, Usmanova G. Sulfuric acid assisted extraction and fractionation of porphyrins from heavy petroleum residuals with a high content of vanadium and nickel. Petroleum Science and Technology.

2015;**33**:992-998. DOI: 10.1080/10916466.2015.1030078

1999;**52**:381-495

DOI: 10.1021/ef201819j

168 Phthalocyanines and Some Current Applications

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/67882

#### **Abstract**

The purpose of this chapter is the brief review of the fundamental study of porphyrin "theranostics" by DNA. Porphyrins have been studied as photosensitizer for photody‐ namic cancer therapy. The activity control of fluorescence emission and photosensitized singlet oxygen generation by porphyrins using the interaction with DNA is the initial step in achieving theranostics. To control these photochemical activities, several types of electron donor‒connecting porphyrins were designed and synthesized. The theoretical calculations speculated that the photoexcited state of these porphyrins can be deactivated via intramolecular electron transfer, forming a charge‒transfer state. The electrostatic interaction between the cationic porphyrin and DNA predicts a rise in the energy of the charge‒transfer state, leading to the inhibition of electron transfer quenching. Pyrene‒ and anthracene‒connecting porphyrins showed almost no fluorescence in an aqueous solution. Furthermore, these porphyrins could not photosensitize singlet oxygen gen‐ eration. These porphyrins bind to a DNA groove through an electrostatic interaction, resulting in the increase of fluorescence intensity. The photosensitized singlet oxygen‒ generation activity of DNA‒binding porphyrins could also be confirmed. On the other hand, several other porphyrins could not demonstrate the activity control properties. To realize effective activity control, a driving force of more than 0.3 eV is required for the porphyrins.

**Keywords:** cationic porphyrin, DNA, singlet oxygen, electron transfer, fluorescence

© 2017 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**

"Theranostics" [1–3] is a relatively new technical term that includes the meanings of ther‐ apeutics and diagnostics [4–9]. The purpose of this review is an introduction of examples of theranostics using porphyrins. Porphyrins can emit relatively strong fluorescence in the wavelength range of visible light and generate singlet oxygen (1 O2 ), an important reactive oxygen species [10]. Singlet oxygen is generated through energy transfer from the triplet excited (T1 ) state of the photosensitizer to the ground state of oxygen molecules (<sup>3</sup> O2 ) [11–13]. Fluorescence imaging is the fundamental mechanism of photodynamic diagnosis (PDD) [14], and 1 O2 is the important reactive species for photodynamic therapy (PDT) [15]. PDT is a less‐ invasive and promising treatment for cancer and other nonmalignant conditions [4–9, 15]. In general, a mechanism of PDT is the oxidation of biomacromolecules, including DNA and proteins, by 1 O2 , which is generated through energy transfer from the excited photosensitizer to oxygen molecules. Porphyrins have been extensively studied as photosensitizers of PDT. Porfimer sodium [16] and talaporfin sodium [17] are especially important clinical drugs used in PDT (**Figure 1**). The control of the photoexcited state of porphyrins by targeting molecules or surrounding environments is the fundamental mechanism of theranostics. In this chapter, the fundamental studies about DNA‐targeting porphyrin theranostics are introduced. DNA is a potentially important target molecule of PDT. Indeed, many DNA‐targeting drugs have been studied and reported [18–20].

**Figure 1.** Structures of examples of PDT photosensitizers, porfimer sodium and talaporfin sodium.

#### **1.1. Photodynamic therapy**

**1. Introduction**

170 Phthalocyanines and Some Current Applications

excited (T1

proteins, by 1

O2

been studied and reported [18–20].

and 1 O2

"Theranostics" [1–3] is a relatively new technical term that includes the meanings of ther‐ apeutics and diagnostics [4–9]. The purpose of this review is an introduction of examples of theranostics using porphyrins. Porphyrins can emit relatively strong fluorescence in the

oxygen species [10]. Singlet oxygen is generated through energy transfer from the triplet

Fluorescence imaging is the fundamental mechanism of photodynamic diagnosis (PDD) [14],

to oxygen molecules. Porphyrins have been extensively studied as photosensitizers of PDT. Porfimer sodium [16] and talaporfin sodium [17] are especially important clinical drugs used in PDT (**Figure 1**). The control of the photoexcited state of porphyrins by targeting molecules or surrounding environments is the fundamental mechanism of theranostics. In this chapter, the fundamental studies about DNA‐targeting porphyrin theranostics are introduced. DNA is a potentially important target molecule of PDT. Indeed, many DNA‐targeting drugs have

**Figure 1.** Structures of examples of PDT photosensitizers, porfimer sodium and talaporfin sodium.

 is the important reactive species for photodynamic therapy (PDT) [15]. PDT is a less‐ invasive and promising treatment for cancer and other nonmalignant conditions [4–9, 15]. In general, a mechanism of PDT is the oxidation of biomacromolecules, including DNA and

, which is generated through energy transfer from the excited photosensitizer

) state of the photosensitizer to the ground state of oxygen molecules (<sup>3</sup>

O2

), an important reactive

O2

) [11–13].

wavelength range of visible light and generate singlet oxygen (1

Photodynamic therapy is a promising and less‐invasive treatment for cancer [4‐9, 15]. Por‐ phyrins are used as photosensitizers of PDT (**Figure 2**). The abovementioned porphyrins, porfimer sodium [16] and talaporfin sodium [17], are especially important photosensitizers. Under visible light irradiation, especially long wavelength visible light (wavelength > 650 nm), an administered porphyrin photosensitizer generates 1 O2 through energy transfer to an oxy‐ gen molecule (the type II mechanism) [21]. Since human tissue has a relatively high transpar‐ ency for visible light, especially red light, visible light rarely demonstrates side effects. Critical targets of 1 O2 include mitochondria and enzyme proteins; DNA is also an important target for PDT [22–26]. In general, the 1 ∑g + state of 1 O2 (1 O2 ( 1 ∑g + )) is mainly formed through the energy transfer from the T1 state of photosensitizers. This state of 1 O2 has relatively high energy, about 1.6 eV, corresponding to the ground state; however, the lifetime is very short (several picosec‐ onds). The 1 O2 ( 1 ∑g + ) is rapidly converted to the 1 Δg state (1 O2 ( 1 Δg )), which has a relatively long lifetime (several microseconds). Therefore, 1 O2 ( 1 Δg ) is a more important reactive oxygen spe‐ cies of PDT. In this chapter, 1 O2 indicates 1 O2 ( 1 Δg ) without explanation. This biomacromolecule damage induces apoptosis and/or necrosis. Apoptosis, a programed death of cancer cells, is considered the main mechanism of PDT [15, 27]. Necrosis also contributes to the mechanism of cell death in the case of severe damage of biomacromolecules by a high dose of photosen‐ sitizers and intense photoirradiation [15]. In the case of DNA‐targeting PDT, 1 O2 selectively oxidizes guanines. The main oxidized product of guanine is 8‐oxo‐7,8‐dihydrodeoxyguanine [28–30].

#### **1.2. Aminolevulinic acid**

One of the most important practical applications of theranostics is the method using the administration of 5‐aminolevulinic acid (5‐ALA, see **Figure 3**) [31–33]. Although the strategy of 5‐ALA theranostics is different from the activity control of the photosensitizer by target molecules mentioned in this chapter, this method is important for cancer theranostics. 5‐ ALA is the source of protoporphyin IX (PPIX) in human cells. In the normal cell, PPIX is converted into iron porphyrin, which cannot emit fluorescence. However, in cancer cells,

**Figure 2.** A general procedure of PDT.

**Figure 3.** PPIX formation from 5‐ALA.

PPIX is selectively concentrated. Several mechanisms for this cancer‐selective concentration of PPIX have been speculated [34, 35]. Because PPIX demonstrates relatively strong red fluo‐ rescence around 650 nm and under blue light irradiation around 450 nm, this phenomenon can be applied to cancer diagnosis. Indeed, the diagnosis of 5‐ALA is clinically applied to the treatment of cancer, for example, malignant brain tumors [36, 37] and bladder cancer [38]. Furthermore, PPIX can photosensitize 1 O2 generation. Although the efficiency of <sup>1</sup> O2 generation by free PPIX is relatively low, the 1 O2 ‐generating activity of PPIX can be increased depending on the environment [39]. These properties of 5‐ALA and PPIX can be used in cancer theranostics.

#### **1.3. Strategy of porphyrin theranostics with target biomolecules**

**Figure 4** shows the energy diagram of the relaxation process of photoexcited porphyrins and theranostics. The singlet excited (S1 ) state of the photosensitizer (Sens\*(S1 )) is formed by photoirradiation. In the OFF state, without the target biomacromolecules, the S<sup>1</sup> state is rap‐ idly quenched, and the excitation energy is dispersed as heat. For example, intramolecular

**Figure 4.** An energy diagram of the relaxation process of photoexcited porphyrin.

electron transfer is a convenient pathway for the quenching to control photochemical activity. In the presence of target molecules, the interaction between the photosensitizer (Sens) and the target molecule inhibits the intramolecular electron transfer. The S1 state with target mol‐ ecules can emit fluorescence (ON state). In the case of porphyrin, the quantum yield of fluo‐ rescence (Φ<sup>f</sup> ) is almost 10% for a relatively intense case. In addition, the intersystem crossing proceeds with a relatively large quantum yield (ΦT); more than 50% is a sufficient value for the Φ<sup>T</sup> . These processes are expressed by the following equations:

$$\mathbf{Sens} \star \mathbf{hv} \quad \twoheadrightarrow \mathbf{Sens}^\*(\mathbf{S}\_1) \tag{1}$$

$$\text{Sens}^\*(\mathcal{S}\_1) \to \text{Sens} \text{ } \text{\textquotedblleft heat \left(Activity . OFF\right)}\tag{2}$$

$$\text{Sens}^\*(\text{S}\_1) \to \text{Sens} \text{} \text{} \text{} \text{hv} \left( \text{Activity:ON} \right) \tag{3}$$

$$\text{Sens}^\*(\text{S}\_\text{i}) \to \text{Sens}^\*(\text{T}\_\text{i}) \text{(Activity:ON)}\tag{4}$$

$$\text{Sens}^\*(\text{T}\_1) + \text{\textquotedblleft O}\_2 \rightarrow \text{\textquotedblright}\text{O}\_2 \{ \text{Activity:ON} \}\tag{5}$$

where Sens\*(T1 ) is the T1 state of the photosensitizer. **Figure 5** shows the scheme of the activity control of photosensitizer by DNA. In the case of DNA, several forms of the binding interac‐ tion can be speculated [40–43]. For example, an electrostatic interaction can switch the activity of photosensitizers.

PPIX is selectively concentrated. Several mechanisms for this cancer‐selective concentration of PPIX have been speculated [34, 35]. Because PPIX demonstrates relatively strong red fluo‐ rescence around 650 nm and under blue light irradiation around 450 nm, this phenomenon can be applied to cancer diagnosis. Indeed, the diagnosis of 5‐ALA is clinically applied to the treatment of cancer, for example, malignant brain tumors [36, 37] and bladder cancer

O2

O2

depending on the environment [39]. These properties of 5‐ALA and PPIX can be used in

**Figure 4** shows the energy diagram of the relaxation process of photoexcited porphyrins

idly quenched, and the excitation energy is dispersed as heat. For example, intramolecular

photoirradiation. In the OFF state, without the target biomacromolecules, the S<sup>1</sup>

) state of the photosensitizer (Sens\*(S1

generation. Although the efficiency of <sup>1</sup>

‐generating activity of PPIX can be increased

O2

)) is formed by

state is rap‐

[38]. Furthermore, PPIX can photosensitize 1

generation by free PPIX is relatively low, the 1

and theranostics. The singlet excited (S1

**1.3. Strategy of porphyrin theranostics with target biomolecules**

**Figure 4.** An energy diagram of the relaxation process of photoexcited porphyrin.

cancer theranostics.

**Figure 3.** PPIX formation from 5‐ALA.

172 Phthalocyanines and Some Current Applications

**Figure 5.** Scheme of the binding interaction between photosensitizers and DNA and the activity switching of photosensitizers through the interaction with DNA.

#### **2. Control of fluorescence and <sup>1</sup> O2 ‐generating activity of alkaloids by DNA**

Photosensitized DNA damage is an important process in medical applications of photochemi‐ cal reactions [44, 45]. In this section, the activity control of naturally occurring photosensitizers

**Figure 6.** Structures of berberine (left) and palmatine (right).

is introduced. Berberine and palmatine are alkaloids (**Figure 6**). These molecules barely emit fluorescence. The S<sup>1</sup> state of these alkaloids deactivates within 40~50 ps through intramolecu‐ lar electron transfer in aqueous solution [46–48]. Since these alkaloids are cationic compounds, in the presence of DNA, an anionic polymer, berberine and palmatine spontaneously bind to the DNA strand through electrostatic interaction. Indeed, it was reported that berberine pref‐ erentially binds to adenine‐thymine–rich minor grooves [49]. The minor groove bindings of berberine and palmatine could be speculated from molecular mechanics calculation [48]. The interaction between these alkaloids and DNA was investigated using oligonucleotides of the adenine‐thymine sequence (AATT: d(AAAATTTTAAAATTTT)<sup>2</sup> ) and the guanine‐containing sequence (AGTC: d(AAGCTTTGCAAAGCTT)<sup>2</sup> ) [48]. The apparent binding constant can be easily estimated from the absorption spectral change of these alkaloids, and the reported val‐ ues are relatively high [48]. The fluorescence intensity of berberine and palmatine was mark‐ edly increased in the presence of DNA. The Ф<sup>f</sup> and the fluorescence lifetimes (*τ*<sup>f</sup> ) of berberine and palmatine were markedly increased through interaction with DNA (**Table 1**).

Furthermore, the 1 O2 ‐generation activity of berberine and palmatine was markedly enhanced by DNA. In aqueous solution, berberine and palmatine hardly photosensitize <sup>1</sup> O2 generation.


The fluorescence properties were examined in a 10‐mM sodium phosphate buffer (pH = 7.6). The ФΔ values were determined in deuterium oxide. These values were reported in the literature [48].

**Table 1.** Fluorescence and photosensitized 1 O2 ‐generating activities of berberine and palmatine in the absence or presence of DNA.

However, in the presence of DNA, the near‐infrared emission at around 1270 nm, assigned to the radiative deactivation of 1 O2 into its ground state, was clearly observed under pho‐ toirradiation of these alkaloids. The estimated quantum yield of <sup>1</sup> O2 generation (ФΔ) using the reference compound, methylene blue (ФΔ = 0.52) [50], depended on the sequence and decreased for the guanine‐containing sequence (**Table 1**). These characteristics are the funda‐ mental mechanisms of theranostics. The theranostics mechanism of berberine and palmatine can be explained as follows:


is introduced. Berberine and palmatine are alkaloids (**Figure 6**). These molecules barely emit

lar electron transfer in aqueous solution [46–48]. Since these alkaloids are cationic compounds, in the presence of DNA, an anionic polymer, berberine and palmatine spontaneously bind to the DNA strand through electrostatic interaction. Indeed, it was reported that berberine pref‐ erentially binds to adenine‐thymine–rich minor grooves [49]. The minor groove bindings of berberine and palmatine could be speculated from molecular mechanics calculation [48]. The interaction between these alkaloids and DNA was investigated using oligonucleotides of the

easily estimated from the absorption spectral change of these alkaloids, and the reported val‐ ues are relatively high [48]. The fluorescence intensity of berberine and palmatine was mark‐

> AATT 0.093 0.30 (0.30) 3.7 (0.42) 11.9 (0.28) 0.066 AGTC 0.043 0.12 (0.60) 1.6 (0.32) 8.0 (0.08) 0.036

> AATT 0.054 0.16 (0.39) 2.3 (0.45) 6.9 (0.16) 0.044 AGTC 0.031 0.14 (0.54) 1.4 (0.37) 5.9 (0.09) 0.030

The fluorescence properties were examined in a 10‐mM sodium phosphate buffer (pH = 7.6). The ФΔ values were

and palmatine were markedly increased through interaction with DNA (**Table 1**).

by DNA. In aqueous solution, berberine and palmatine hardly photosensitize <sup>1</sup>

Berberine Without <0.001 0.05 nd

Palmatine Without <0.001 0.04 nd

determined in deuterium oxide. These values were reported in the literature [48].

O2

adenine‐thymine sequence (AATT: d(AAAATTTTAAAATTTT)<sup>2</sup>

sequence (AGTC: d(AAGCTTTGCAAAGCTT)<sup>2</sup>

**Figure 6.** Structures of berberine (left) and palmatine (right).

174 Phthalocyanines and Some Current Applications

edly increased in the presence of DNA. The Ф<sup>f</sup>

O2

**Alkaloid DNA Ф<sup>f</sup>** *τ<sup>f</sup>*

**Table 1.** Fluorescence and photosensitized 1

presence of DNA.

state of these alkaloids deactivates within 40~50 ps through intramolecu‐

) and the guanine‐containing

O2

) of berberine

generation.

) [48]. The apparent binding constant can be

and the fluorescence lifetimes (*τ*<sup>f</sup>

**/ns (ratio) ФΔ**

‐generating activities of berberine and palmatine in the absence or

‐generation activity of berberine and palmatine was markedly enhanced

fluorescence. The S<sup>1</sup>

Furthermore, the 1

**3.** Fluorescence intensity and the intersystem crossing yield are increased, resulting in the enhancement of energy transfer to the oxygen molecule to generate 1 O2 .

**Figure 7.** Intramolecular electron transfer in the S1 state of berberine and palmatine and the activity switching by DNA.
