**2. Biological applications of porphyrins**

#### **2.1. Porphyrins in photodynamic therapy (PDT)**

#### *2.1.1. A brief historical of PDT*

The term PDT—photodynamic therapy—is recent. However, the heliotherapy—the therapeutic exposure to sunlight—was already used more than 4000 years ago by Egyptians, Greeks, and Indians as a treatment for several skin disorders, like psoriasis, vitiligo, cancer and even psychosis [21–29]. Heliotherapy, recently known as phototherapy, employs either UV and visible light with/without an exogenous photosensitizer. The photosensitizer is a molecule which when exposed to light absorbs determined wavelength becomes electronically excited and starts photochemical reactions that can produce a desirable beneficial effect, as in the case of vitamin D synthesis or damage and death, as in the case of tumor and infections treatment [2, 30]. Phototherapy without an exogenous photosensitizer is used in dermatology to treat vitiligo, eczema, neonatal jaundice and vitamin D deficiency, and even some cancer types [30–33]. During 18th and 19th centuries, phototherapy without exogenous photosensitizer was used in France in the treatment of many diseases, including tuberculosis, rheumatism, edema, rickets and paralysis [28, 34]. When an exogenous photosensitizer is used in tandem with the sunlight, this therapy is called photochemotherapy. An example of the exogenous photosensitizer is the psoralen series (**Figure 3**). These molecules are used as active treatments of HIV-associated dermatoses, seborrheic dermatitis, mycosis fungoids, prurigo, palmar and plantar pustulosis, among other diseases [30, 35]. The use of psoralens and ultraviolet light— UV (300–400 nm) was used by ancient Egyptians to treat vitiligo in the past and has been accepted for the treatment of psoriasis (PUVA) and in immunotherapy throughout the world [22, 27, 30, 35, 36].

Photodynamic therapy (PDT) is a non-invasive treatment method that uses light, photosensitizer and molecular oxygen for the treatments of cancer, inflammation, immunological Free-Base and Metal Complexes of 5,10,15,20-Tetrakis(N-Methyl Pyridinium L)Porphyrin... http://dx.doi.org/10.5772/intechopen.68225 5

**Figure 3.** Psoralen series.

**2. Biological applications of porphyrins**

**Figure 2.** *Ortho, meta* and *para* isomers of MnTMPyP.

4 Phthalocyanines and Some Current Applications

**2.1. Porphyrins in photodynamic therapy (PDT)**

The term PDT—photodynamic therapy—is recent. However, the heliotherapy—the therapeutic exposure to sunlight—was already used more than 4000 years ago by Egyptians, Greeks, and Indians as a treatment for several skin disorders, like psoriasis, vitiligo, cancer and even psychosis [21–29]. Heliotherapy, recently known as phototherapy, employs either UV and visible light with/without an exogenous photosensitizer. The photosensitizer is a molecule which when exposed to light absorbs determined wavelength becomes electronically excited and starts photochemical reactions that can produce a desirable beneficial effect, as in the case of vitamin D synthesis or damage and death, as in the case of tumor and infections treatment [2, 30]. Phototherapy without an exogenous photosensitizer is used in dermatology to treat vitiligo, eczema, neonatal jaundice and vitamin D deficiency, and even some cancer types [30–33]. During 18th and 19th centuries, phototherapy without exogenous photosensitizer was used in France in the treatment of many diseases, including tuberculosis, rheumatism, edema, rickets and paralysis [28, 34]. When an exogenous photosensitizer is used in tandem with the sunlight, this therapy is called photochemotherapy. An example of the exogenous photosensitizer is the psoralen series (**Figure 3**). These molecules are used as active treatments of HIV-associated dermatoses, seborrheic dermatitis, mycosis fungoids, prurigo, palmar and plantar pustulosis, among other diseases [30, 35]. The use of psoralens and ultraviolet light— UV (300–400 nm) was used by ancient Egyptians to treat vitiligo in the past and has been accepted for the treatment of psoriasis (PUVA) and in immunotherapy throughout the world

Photodynamic therapy (PDT) is a non-invasive treatment method that uses light, photosensitizer and molecular oxygen for the treatments of cancer, inflammation, immunological

*2.1.1. A brief historical of PDT*

[22, 27, 30, 35, 36].

diseases and bacterial infections [8, 37–41]. In ancient times, phototherapy was used based on the observation of positive results without a mechanistic knowledge. People using and advocating phototherapy did know the key role of the photosensitizer in this type of treatment. In that times, the photosensitizer role was played by an endogenous biomolecule absorbing sunlight. The domain of the PDT mechanism initiated with the isolation of hematoporphyrin (Hp) (**Figure 4**) [28, 42]. From dried blood cells by Scherer in 1841 followed by the discovery of its fluorescence properties in 1871 [43]. In 1911 and 1913, the side effects of sun exposure after the administration of hematoporphyrin were described by Hausmann and Friedrich Meyer-Bertz. The latter scientist tested on himself the effect of Hp and sun and provided the first scientific communication of human photosensitization [44]. Besides, the powerful cytotoxic effect of phototherapy, another significant finding favoring the consolidation of this type of treatment, was the report of Auler and Banzer showing the affinity of Hp for cancer cells in 1942 [45]. In the following, several other studies led to the development of new range of porphyrinic photosensitizers [28, 43, 46–51].

#### *2.1.2. The PDT mechanism*

The Jablonski diagram [52], first proposed by Professor Alexander Jablonski in 1935, has been used to describe the photodynamic processes of photosensitizer molecules used in PDT. The PDT principles are based on the presence of an endogenous or exogenous photosensitizer in the target tissue that can absorb red light to be promoted to a long-lived electronic excited state. In the electronic excited state, the photosensitizer triggers photooxidative events directly or more commonly via energy transfer to molecular oxygen. The quantum yield triplet state generation depends on the molecular structure, and the energy transfer to molecular oxygen competes with other deactivating routes for the excited state [25].

According to **Figure 5**, Jablonski diagram shows that the ground state photosensitizer (S0 ) can absorb a photon and be converted to the short-lived excited singlet state (Sn) at different

**Figure 4.** Hematoporphyrin.

vibrational sublevels (Sn'). The Sn state, if n > 1 can lose energy *via* internal conversion (IC) to populate the first excited single state (S<sup>1</sup> ). In the first singlet excited state, the photosensitizer can return to the ground state via fluorescence and thermal irradiation. Also, the S<sup>1</sup> state of the photosensitizer can undergo intersystem crossing by spin inversion and populate the lowerenergy first excited triplet state (T<sup>1</sup> ), a long-lived state [2, 30, 37, 49]. At this point, two different reaction processes involving molecular oxygen can occur Type I or Type II processes. In the first process, Type I, the photosensitizer in a triplet excited state is reduced with organic substrates by electron exchange. The reduced photosensitizer can react with molecular oxygen (3 O2 ) to produce reactive oxygen species (ROS) such superoxide anion (O2 − ·), hydroxyl radical (OH·) and hydrogen peroxide (H<sup>2</sup> O2 ) [30, 37, 53]. In the second process, Type II, the triplet excited state photosensitizer transfers energy to molecular oxygen, resulting in a long-lived and highly reactive species, the singlet oxygen (1 O2 ) [37, 49, 54]. Types I and II mechanisms occur concomitantly. However, Type II is the dominant process during PDT [30, 37].

Free-Base and Metal Complexes of 5,10,15,20-Tetrakis(N-Methyl Pyridinium L)Porphyrin... http://dx.doi.org/10.5772/intechopen.68225 7

**Figure 5.** Energy levels of Jablonski diagram for a typical type II photosensitizer and oxygen.

In PDT, singlet oxygen is the principal reactive species. However, as well as others ROS, singlet oxygen has the capacity of damage limited due to its short lifetime (~100 ns in lipid regions of membranes and 250 ns in the cytoplasm) [30, 49, 55], and a diffusion range of approximately 45 nm in the cellular medium [28, 56–58]. The PDT has amino acid residues in proteins, unsaturated lipids, and DNA as the targets for oxidation leading to cell damage [59–61].

#### *2.1.3. Porphyrin as photosensitizers*

vibrational sublevels (Sn'). The Sn state, if n > 1 can lose energy *via* internal conversion (IC) to

photosensitizer can undergo intersystem crossing by spin inversion and populate the lower-

reaction processes involving molecular oxygen can occur Type I or Type II processes. In the first process, Type I, the photosensitizer in a triplet excited state is reduced with organic substrates by electron exchange. The reduced photosensitizer can react with molecular oxygen

excited state photosensitizer transfers energy to molecular oxygen, resulting in a long-lived

occur concomitantly. However, Type II is the dominant process during PDT [30, 37].

O2

can return to the ground state via fluorescence and thermal irradiation. Also, the S<sup>1</sup>

) to produce reactive oxygen species (ROS) such superoxide anion (O2

O2

). In the first singlet excited state, the photosensitizer

−

) [37, 49, 54]. Types I and II mechanisms

), a long-lived state [2, 30, 37, 49]. At this point, two different

) [30, 37, 53]. In the second process, Type II, the triplet

state of the

·), hydroxyl radical

populate the first excited single state (S<sup>1</sup>

energy first excited triplet state (T<sup>1</sup>

6 Phthalocyanines and Some Current Applications

**Figure 4.** Hematoporphyrin.

(OH·) and hydrogen peroxide (H<sup>2</sup>

and highly reactive species, the singlet oxygen (1

(3 O2 An ideal photosensitizer needs to have the following characteristics: (1) chemical purity; (2) high yield of singlet oxygen production; (3) high absorption coefficient in the red region of the visible spectrum (680–800 nm). Wavelengths longer than 900 nm should be avoided due to their insufficient energy to excite a dye photosensitizer to the triplet state; (4) efficient accumulation in tumor tissue associated with a rapid clearance in healthy organs; (5) low toxicity in the dark extensive to their metabolites; and (6) small aggregation [8, 30, 49, 62–64].

Porphyrins satisfy most of the desirable properties of photosensitizers, such as high efficiency of singlet oxygen generation, absorption of the higher wavelengths of the electromagnetic spectrum and a relatively higher affinity for malignant cells. Porphyrins have 18π electrons on the aromatic macrocycle that responds for the "*Soret*" band, with a strong absorption band around 400 nm, and Q bands in the 500–700 nm range that constitute the therapeutic window for this photosensitizer (**Figure 6**) [10, 65]. The absorption spectrum of the porphyrins is influenced by ligands and the central metal [66–68].

**Figure 6.** Porphyrin absorption spectrum. a = Soret band; b = Q band.

In the early twentieth century, data of literature described experiments that demonstrated the potential role of Hp in the detection and treatment of cancers; however, one of the major drawbacks was the large doses required to achieve consistent photosensitizer uptake in tumors, which led to inappropriate phototoxicity [45, 69–71]. In 1955, Schwartz et al. [72] demonstrated Hp to be impure and attributed selective fluorescence of malignant tissue after in vivo administration of Hp to a mixture of porphyrins with different properties. Subsequent studies led to the development of a derivative of hematoporphyrin (HpD) by the treatment of crude Hp with acetic and sulfuric acids, which enhanced tumor accumulation. The ability to accumulate selectively in neoplastic tissue using lower doses of HpD than Hp was reported by Lipson and coworkers [73–77]. In 1972, Diamond et al. demonstrated the destructive potential of HpD irradiated with white light on glioma in rats [78]. Six years later, Dougherty et al. reported the partial and complete response of many tumors, including malignant melanomas and carcinomas of the colon, breast, and prostate, treated by photodynamic therapy using HpD as a photosensitizer [79]. In the following, HpD compounds were purified, many of the less active monomers were removed, and the most efficient HpD derivatives were used to produce Photofrin (**Figure 7**).

For a complete study of different porphyrinic photosensitizers [80–109], it is recommended the reviews Josefsen et al. [2], Connor et al. [25], Pushpan et al. [28], and Ethirajan et al. [49]

Among a diversity of porphyrinic photosensitizers, *meso*-tetraphenylporphyrin (TPP) and TMPyP are readily synthesized and metallized, and several derivatives have been studied as a photosensitizer for PDT. The photochemical efficiency of anionic 5,10,15,20-*meso*-tetra(4-sulfonatophenyl)porphyrin (H2 TPPS4 ) (**Figure 8A**) was compared with *meso*-tetraphenyl porphyrins with a lower number of sulfonate groups [99, 100] and Free-Base and Metal Complexes of 5,10,15,20-Tetrakis(N-Methyl Pyridinium L)Porphyrin... http://dx.doi.org/10.5772/intechopen.68225 9

**Figure 7.** Photofrin.

In the early twentieth century, data of literature described experiments that demonstrated the potential role of Hp in the detection and treatment of cancers; however, one of the major drawbacks was the large doses required to achieve consistent photosensitizer uptake in tumors, which led to inappropriate phototoxicity [45, 69–71]. In 1955, Schwartz et al. [72] demonstrated Hp to be impure and attributed selective fluorescence of malignant tissue after in vivo administration of Hp to a mixture of porphyrins with different properties. Subsequent studies led to the development of a derivative of hematoporphyrin (HpD) by the treatment of crude Hp with acetic and sulfuric acids, which enhanced tumor accumulation. The ability to accumulate selectively in neoplastic tissue using lower doses of HpD than Hp was reported by Lipson and coworkers [73–77]. In 1972, Diamond et al. demonstrated the destructive potential of HpD irradiated with white light on glioma in rats [78]. Six years later, Dougherty et al. reported the partial and complete response of many tumors, including malignant melanomas and carcinomas of the colon, breast, and prostate, treated by photodynamic therapy using HpD as a photosensitizer [79]. In the following, HpD compounds were purified, many of the less active monomers were removed, and the most efficient HpD derivatives were used to produce

For a complete study of different porphyrinic photosensitizers [80–109], it is recommended the reviews Josefsen et al. [2], Connor et al. [25], Pushpan et al. [28], and Ethirajan et al. [49]

Among a diversity of porphyrinic photosensitizers, *meso*-tetraphenylporphyrin (TPP) and TMPyP are readily synthesized and metallized, and several derivatives have been studied as a photosensitizer for PDT. The photochemical efficiency of anionic

with *meso*-tetraphenyl porphyrins with a lower number of sulfonate groups [99, 100] and

TPPS4

) (**Figure 8A**) was compared

5,10,15,20-*meso*-tetra(4-sulfonatophenyl)porphyrin (H2

**Figure 6.** Porphyrin absorption spectrum. a = Soret band; b = Q band.

8 Phthalocyanines and Some Current Applications

Photofrin (**Figure 7**).

with 5,10,15,20-tetrakis(4-sulfonatophenyl-21,23-dichalcogenaporphyrin [110] (**Figure 8B**). These studies showed that H2 TPPS4 is less efficient in PDT than *meso*-tetraphenyl porphyrins with a lower number of sulfonate groups. Also, the replacement of nitrogen atoms of the macrocycle by chalcogens S and Se increased the photodynamic efficiency of the porphyrin in vitro and in vivo studies. Particularly in vivo, these chalcogen derivatives exhibited lower toxicity, morbidity and side effects post administration in animal models.

Regarding TMPyP, the focus of the present study, its efficiency as a photosensitizer is related to its topology. A study comparing photodamage in a mitochondrial membrane model modulated by the topology of TPPS4 and 5,10,15,20-tetrakis(N-methyl pyridinium L)porphyrin (TMPyP) [8] shows that in L-α-phosphatidylcholine/cardiolipin (PC/CL)liposomes (mitochondrial membrane model) both porphyrin can damage the membrane *via* the Type II mechanism. However, the injuries on the lipid membranes promoted by TMPyP were greater than the damages promoted by TPPS4 due to the affinity between TMPyP and this biological

**Figure 8.** TPP-based photosensitizers. (A) Tetrasulfonated *meso*-tetraphenyl porphyrin; (B) *meso*-tetrakis(4-sulfona tophenyl)-21,23-dichalcogenaporphyrin.

structure [111, 112] that in turn influences the photosensitizer and the generation of longlived singlet oxygen. In cells, the positively charged TMPyP accumulates in the nucleus and mitochondria and could attack DNA, mitochondrial DNA and cardiolipin. The association of TMPyP with the inner mitochondrial membranes due to the affinity to cardiolipin favors the generation of singlet oxygen in situ with a high efficiency since its concentration is higher in the hydrophobic core of the lipid bilayers. Metalloporphyrins have also been studied as potential sensitizers for PDT. However, the results were less promising than those obtained with the free-base species [113, 114].

#### **2.2. Porphyrins in chemical therapy**

The synthetic analogs of porphyrins are widely used in therapy of diseases connected to oxidative stress processes. A quantitative structure-activity relationship (QSAR) studies have been performed to identify the optimal active molecule within a series of analog structure characteristics to diversify the biological action of the compound. The QSAR studies can correlate the physicochemical characteristics that affect the compound's activity in biological systems. These studies assumed that the binding affinity of the compound to the target receptor could determinate the biological activity [115]. The biological effects of two meso-tetrakis porphyrins, TPPS4 (anionic) and TMPyP (cationic) demonstrated that the cationic porphyrin has affinity to the inner mitochondrial membrane [99]. Therefore, in mitochondria, Mn3+TMPyP has been used as an antioxidant against superoxide ions. The replacement of manganese by an iron ion in TMPyP makes this porphyrin a prooxidant agent [116]. Au-porphyrins have been reported as excellent antiproliferative agents, showing cytotoxic effects on cancer cells. Regarding to the mimetic SOD activity of porphyrins, the correlation between the metal-centered reduction potential and the catalytic rate constant for the O2 •− dismutation was found for Fe and Mn porphyrins. The structure-activity relationships have been established over the years by the rate-limiting step of metal reduction of this class of compounds [117]. Modulation of SOD activity has been achieved by decreasing the electron density of the groups at the meso and β-pyrrile positions, thus increasing the Mn3+/Mn2+ potential and facilitating its reduction [118–120]. Either the mimetic SOD activity can occur when the O2 · − is directed to the catalytic site by the metal-centered positive charges via electrostatic facilitation [118, 119]. The manganese (III) 5,10,15,20-tetrakis(N-ethylpyridinium-2-yl) porphyrin (Mn3+TE-2-PyP5+, E½ = +228 mV *vs* NHE, log kcat = 7.76) and manganese (III) 5,10,15,20-tetrakis(N-n-hexylpyridinium-2-yl) porphyrin (MnTnHex-2-PyP5+, E½ = +314 mV *vs* NHE, log kcat = 7.48), alkylated manganese (III) 5,10,15,20-tetrakis(2-pyridyl)porphyrin (MnT-2-Pyp<sup>+</sup> ), combined the thermodynamic and electrostatic optimizations and yielded compounds because they exhibit the E½ close to the reduction potential of the SOD enzyme and are excellent mimetics of the SOD activity (E½ ≅ +300 mV *vs* NHE, kcat ≅ 2 × 10<sup>9</sup> M−1 s−1) [19, 117–122]. Recently, it has been reported that the para isomer (E½ = +60 mV *vs* NHE) of Mn3+TMPyP is less efficient as a SOD mimic relative than the *ortho* isomer (E½ = +260 mV *vs* NHE) [12, 19, 123, 124].

In a cell redox balance, the association of Mn3+TMPyP to membrane lipid bilayers can be intrinsically related to the redox potential of the Mn2+/Mn3+ couple. In homogeneous systems, Batinić-Haberle et al. [19] had reported the effect of Mn3+TMPyP in a CL-containing inner mitochondrial membrane under pH 11 to 7.8 conditions. The potential values of Mn2+/Mn3+ redox process were found to be E1/2 =94 mV for *ortho* Mn3+TMPyP and E1/2 = 42 and 50 mV, respectively, for *meta* and *para* isomers. However, in a heterogeneous system, Araujo-Chaves et al. [20] have reported that the *para* isomer has the redox potential increased by the association with the negatively charged interface of lipid bilayers. Interestingly, the association of *para* Mn3+TMPyP to PC/PS liposomes at physiological pH exhibited a redox potential of +110 mV *vs* NHE. The shift of the Mn2+/Mn3+ E1/2 value to a more positive value favors the SOD and peroxidase activities. Theoretical calculations corroborated with these results.
