Photosensitization Reactions In Vitro and In Vivo

Authors


Corresponding authors email: agreer@brooklyn.cuny.edu (Alexander Greer)

Abstract

This review of Photochemistry and Photobiology summarizes articles published in 2010, and highlights progress in the area of photosensitization. The synthesis of conjugated photosensitizers is an area of interest where increasing water solubility has been a goal. Targeting infrared sensitizer absorption has been another goal, and relates to the practical need of deep tissue absorption of light. Photodynamic techniques for inactivating microbes and destroying tumors have been particularly successful. Biologically, singlet oxygen [1O2(1Δg)] is an integral species in many of these reactions, although photosensitized oxidations tuned to electron and hydrogen transfer (Type I) give rise to other reactive species, such as superoxide and hydrogen peroxide. How photoprotection against yellowing, oxygenation and degradation occurs was also an area of topical interest.

This review is only a sampling of papers published in 2010 in Photochemistry and Photobiology covering the area of photosensitization, wishing to highlight readers on some of the advancements that were made. The review has four sections: photosensitizer design; supports for or encapsulation of photosensitizers; photooxidation and photoprotective reactions and photodynamic action. A summary table shows photophysical data of the sensitizers and trapping agents (Table 1).

Table 1.   Photophysical data of the sensitizers and trapping agents.
Compoundλmax (nm)λem (nm)Luminescence lifetime (τ, μs)Fluorescence quantum yield (ΦF)Singlet oxygen quantum yield (ΦΔ)Total (kT) and chemical (kr) quenching rate constants of 1O2 (m−1 s−1)Photodegradation quantum yieldRef.Comments
3565 (CH3OH)2 
4530 (CH3OH)  
5554 (CH3CN)24 (N2) 0.33 (air)0.38 (CH3CN)0.95 ± 0.05 (CD3CN)
0.27 ± 0.03 (CD2Cl2)
kT = 5.54 ± 0.03 × 1073Methylene blue (MB) ΦF = 0.57 (CD3CN) and C60ΦF = 1.00 (CD2Cl2) was used as reference
6559 (CH3CN)1.38 (N2) <0.1 (air)0.021 (CH3CN)0.42 ± 0.03 (CD3CN)
0.15 ± 0.03 (CD2Cl2)
kT = 0.91 ± 0.11 × 107 
7525 (DMSO)4Sizable absorption peaks were located at 645 and 685 nm
8460, 580 near equivalent (CHCl3)1.0 (C6H6)5Hypocrellin B ΦΔ = 0.76 was used as reference
9526 (CHCl3)0.93 (C6H6) 
10656 (DMF)661 (DMF) λex = 375 nm0.058 (DMF)0.196 (DMF)
0.726 (DMF/HCl)
6ZnPc ΦF = 0.20 (pyridine) and ΦΔ = 0.56 (DMF) was used as reference. [HCl]  = 5 × 10−4 m
11748 (DMF)751 (DMF) λex = 375 nm0.027 (DMF)0.754 (DMF)
0.677 (DMF/HCl)
 
12629 (CH2Cl2)462 (CH2Cl2) λex = 446 nm 727 (CH2Cl2) λex = 702 nm0.09 (CH2Cl2)0.074 ± 0.001 (CH3OH)
1.00 ± 0.025 (PC liposome)
7The phosphatidyl choline (PC) liposome binding constant Kb was 26.8 ± 1.3 (mg/mL)−1
13ca 660 Q-band region8Absorption spectra in LDL, HDL or DMSO solutions
14ca 685 Q-band region 
16420 Soret 514
Qy(1–0) (toluene)
652 (toluene) Q(0–0)
718 (toluene) Q(0–1)
0.086 (toluene)0.60 (toluene)4.65 × 10−4 (azobenzene)9Triplet lifetimes were 44.9 ± 1.0 μs (N2) and 320.6 ± 12.8 ns (O2)
Photodegredation quantum yield was measured with 5,10,15-20-tetraphenylporphyrin ΦF = 0.11 (toluene) as a reference
17678 Q(0,0) (toluene)0.33 ± 0.04 (toluene)
0.20 ± 0.04 (Cremophor RH 40)
0.34 ± 0.07 (Lutrol F 127–Cremophor RH 40)
0.69 ± 0.13 (toluene)
0.40 ± 0.10 (Cremophor RH 40)
0.60 ± 0.10 (Lutrol F 127–Cremophor RH 40)
14Hydroxyaluminum tricarboxymonoamidephthalo-cyanine (AlTcPc) ΦF = 0.42 (DMSO) was used as reference in toluene. Cresyl violet perchlorate (CV) ΦF = 0.60 (cellulose) was used as reference in the gel samples. MB ΦΔ = 0.56 (DMF) was used as reference. The hydrophilic gel was Cremophor® RH40 25% wt/wt. The lipophilic gel was Lutrol® F127 15%, Cremophor® RH40 10% and polypropylene glycol 15%
18676 Q(0,0) (toluene)0.31 ± 0.03 (toluene)
0.21 ± 0.04 (Cremophor RH 40)
0.35 ± 0.06 (Lutrol F 127–Cremophor RH 40)
0.66 ± 0.13 (toluene)
0.60 ± 0.10 (Cremophor RH 40)
0.60 ± 0.10 (Lutrol F 127–Cremophor RH 40)
 
19673 Q(0,0) (THF)0.28 ± 0.06 (THF)
0.02 ± 0.06 (Cremophor RH 40)
0.06 ± 0.03 (Lutrol F 127–Cremophor RH 40)
0.70 ± 0.10 (THF)
0.13 ± 0.06 (Cremophor RH 40)
0.20 ± 0.05 (Lutrol F 127–Cremophor RH 40)
 
20704 Q(0,0) (THF)0.26 ± 0.03 (THF)
0.02 ± 0.05 (Cremophor RH 40)
0.04 ± 0.03 (Lutrol F 127–Cremophor RH 40)
0.69 ± 0.05 (THF)
0.14 ± 0.05 (Cremophor RH 40)
0.26 ± 0.05 (Lutrol F 127–Cremophor RH 40)
 
21340 (H2O)15 
22311 (H2O)390 (H2O) λex = 310 nm  
23350 (H2O) λex = 338 nm
350 (H2O, 1 (mg/mL)−1 DOPC) λex = 338 nm
kT = 0.94 ± 0.09 × 108 (D2O/H2O)
kr = 0.23 ± 0.02 × 108 (D2O/H2O)
kT = 1.7 ± 0.2 × 108 [1 (mg/mL)−1 DOPC]
kr = 0.2 ± 0.2 × 108 [1 (mg/mL)−1 DOPC]
16D2O/H2O mixtures were used. 1,2-di-oleoyl-glycero-3-phosphatidylcholine (DOPC) large unilamellar vesicles. The rate constants were “apparent”kT and kr quenching values
24355 (H2O) λex = 338 nm
338 (H2O, 1 (mg/mL) DOPC) λex = 338 nm
kT = 1.8 ± 0.2 × 108 (D2O/H2O)
kr = 0.22 ± 0.02 × 108 (D2O/H2O)
kT = 2.6 ± 0.2 × 108 [1 (mg/mL)−1 DOPC]
kr = 0.56 ± 0.05 × 108 [1 (mg/mL)−1 DOPC]
 
27232 (H2O)0.014 (1.27 mm O2) 0.018 (0.27 mm O2) 0.027 (<50 μm O2)18Photodegradation quantum yields were measured at λex = 254 nm in H2O (pH = 5.4)
28kT = 0.031 × 108 (CH3OH)
kr = 0.011 × 108 (CH3OH)
20CH3OH solutions contained riboflavin and 10 mm KOH
29kT = 0.39 × 108 (CH3OH)
kr = 0.3 × 108 (CH3OH)
 
30kT = 0.009 × 108 (CH3OH)
kr = 0.001 × 108 (CH3OH)
 
31ca 390 (CH3CN)kT = 4.8 ± 0.1 × 108 (D2O buffer, pD ca 7.2)25Alkaline D2O experiment assumed 33 to be fully ionized
32ca 390 (CH3CN)kT = 0.01 × 108 (with perinaphthenone in D2O) 
33ca 360 (CH3CN)kT = 0.3 ± 0.1 × 108 (D2O buffer, pD ca 7.2)
kT = 7.9 ± 0.2 × 108 (alkaline D2O)
kT = 0.01 × 108 (CH3CN)
 
34ca 390 (CH3CN)kT = 0.3 ± 0.05 × 108 (with MB in D2O) 

Photosensitizer design

The synthesis of conjugated photosensitizers represents an area that is of interest to photochemists where increasing water solubility has been a goal. As would be expected, the water solubility of C60-d-glucose (1) and C60-maltohexaose (2) photosensitizers was enhanced compared to C60 itself (Scheme 1) (1). Upon irradiation with UVA light, the cytotoxicity of 1 and 2 against several cancer cell lines was attributed to singlet oxygen. A Rose Bengal-linker-penicillanic acid (3) and a Rose Bengal-linker-kanamycin conjugate (4) were found to be bactericidal agents under white light irradiation; they led to the eradication of Staphylococcus aureus and Escherichia coli, respectively, under conditions where Rose Bengal and kanamycin individually were ineffective (2). Cyclometalated complexes included a [Pt(Thpy)(HThpy)pyridine]+ (5) and a thienyl/pyridyl Pt(II) butoxycalix[4]arene (6) (3), where O2 quenched the metal-to-ligand charge transfer excited states producing 1O2 in high yields. In CD3CN, the 1O2 quantum yield (ΦΔ) of 5 approached or exceeded 0.95 (Table 1). Sensitizer 5 was examined and was shown to be photocytotoxic to HeLa cells.

Figure Scheme 1..

Targeting redshifted sensitizer absorption has been a goal, and relates to practical needs such as deep tissue absorption of light. Hypericin hydroquinone (7) was redshifted compared to hypericin itself, and exhibited photocytotoxicity on DU145 human prostate carcinoma cells (Scheme 2) (4). One problem was the stability; there was the tendency of the hydroxy groups of 7 to revert to the keto form of hypericin. Somewhat structurally similar to hypericins are the hypocrellins. Sodium hypocrellin B-aminododecanoate (8) and sodium hypocrellin B-dimercaptoundecanoate (9) were synthesized (5), and had surfactant-like properties, arguably more amphiphilically compatible to blood plasma for cellular uptake. Sensitizer 9 was active against human breast carcinoma MCF-7 cells, where singlet oxygen was formed; however, under anoxic conditions species such as the semiquinone radical anion were formed.

Figure Scheme 2..

Water-soluble Zn-azaphthalocyanine (10) and Zn-azanaphthalocyanine (11) were synthesized, each carrying eight quaternary ammonium groups (Scheme 3) (6). Sensitizers 10 and 11 absorbed in the red, had high singlet oxygen quantum yields (ΦΔ), and localized within the lysosomes of Hep2 cells. Other phthalocyanines studied include a series of chiral bis-acetal porphyrazine derivatives, synthesized for absorption and emission in the red (7). The phthalocyanines took the form M[pz(A4-nBn)], in which the pyrrole ring contained A = (2R,3R) 2,3-dimethyl-2,3-dimethoxy-1,4-diox-2-ene, B = β,β′-diisopropoxybenzo, M is Zn ion or two hydrogen atoms and n = 0, 1, 2 (-cis/-trans) or 3. Of the series, porphyrazine H2[pz(trans-A2B2)] (12) performed the best, with a dual function of high phototoxicity and strong fluorescence upon uptake by cells.

Figure Scheme 3..

Zn- and Pd-bacteriopheophorbide (13 and 14), and the water-soluble Pd-bacteriopheophorbide (15) were studied with near-IR radiation in human blood plasma (Scheme 4) (8). The range of photodynamic damage was suggested to be extended by the formation of peroxides via pigment-loaded LDL and HDL. Pd-bacteriopheophorbide 14 had greater stability than 13 and 15, and also produced more reactive oxygen species (ROS) thereby generating hydroperoxides. Another porphyrin system was studied, namely 5,10,15,20-tetra-(phenoxy-3-carbonyl-1-aminonaphthyl)-porphyrin (16), which was synthesized by a naphthylisocyanate condensation reaction (9). The absorption of the 4th Q-band extended out to ca 670 nm, while the triplet porphyrin and singlet oxygen quantum yields were found to be enhanced compared to the parent 5,10,15,20-tetra-(3-hydroxyphenyl)-porphyrin. Sensitizer 16 was also found to be photoactive against human carcinoma HT-29 cells.

Figure Scheme 4..

Supports for or encapsulation of photosensitizers

In addition to free sensitizers, researchers have been active in attaching or enclosing sensitizers by way of heterogeneous platforms and phases. Among other things, highly hydrophobic sensitizers can be solubilized via encapsulation in vesicles, liposomes or capsules for use in aqueous media.

Supports

The photolysis of protoporphyrin IX-supported silica nanoparticles sized 10, 25 or 60 nm led to the formation of singlet oxygen, which could diffuse out of the porous matrix (10). The short diffusion distance of singlet oxygen away from the nanoparticles (ca 20–150 nm) did not inhibit its ability to cause cell death in HCT 116 and HT-29 (colon cancer cells), MCF7 and MDA-MB-231 (breast cancer cells), A431 (epidermoid cells) and LLBC37 (lymphoblastoid cells) and tumor destruction in mice.

The use of quantum dots as photosensitizers dates back only several years. CdTe quantum dots coated with thioglycolic acid sized ca 3.5 nm served as a unique photosensitizer capable of photodestruction of nasopharyngeal carcinoma cells (11). Low concentrations of singlet oxygen were found and Type-I photooxidations produced ROS that arose from the initial production of surface electrons.

Photooxidation mechanisms of DNA are difficult to study in biological systems. Making inroads to understanding the behavior of DNA damage, a photosensitizer [benzo[a]pyrene 7,8-diol 9,10-epoxide N2-guanine (G1Py)] was incorporated into the oligonucleotide 5′-d(CATG1PyCG2TCCTAC) to examine hole injection and migration. To appreciate the oxidative damage, an analysis was done of the Py-modified base, dG1Py and the guanine residue G2 (12). The 355 nm irradiation of the pyrene sensitizer generated a Py•+ by a two-photon process, as well as hydrated electrons that were trapped by O2 to form superoxide. An M+16 nucleoside oxidation product was formed from the photosensitizing dG1Py moiety in which the cyclohexenyl ring opened with a carbonyl group at the rupture site. 18O labeling studies showed that the oxygen atom originated from H218O and not from 18O2.

Container systems and gels

Apoferritin was fragmented and found to self-reassemble and encapsulate methylene blue (13). The apoferritin container with methylene blue guests was cytotoxic against MCF-7 human breast adenocarcinoma cells with 633 nm light and activated singlet oxygen sensor green, a probe which has attracted attention recently due to its high fluorescence intensity in the presence of singlet oxygen. One study was concerned with topical photodynamic therapy applications and reducing sensitizer aggregation. Hydrophilic and lipophilic (Cremophor and Lutrol) gel formulations of tetra-t-butylphthalocyaninato zinc(II) (17), tetrakis(1,1-dimethyl-2-phthalimido)ethylphthalocyaninatozinc(II) (18), 2,3,9,10,16,17,23,24-octakis(decyloxy)phthalocyaninatozinc(II) (19) and 2,3,9,10,16,17,23,24-octakis[(N,N-dimethylamino)ethylsulfanyl]phthalocyaninatozinc(II) (20) were found to generate singlet molecular oxygen with ΦΔ values ranging from 0.20 to 0.60 assessed with imidazole and the bleaching of N,N-diethyl-4-nitrosoaniline (Scheme 5) (14). When accumulated in the lipophilic gel, 17 and 18 bearing branched substituents were less aggregated than 19 and 20 bearing long-chain substituents.

Figure Scheme 5..

Photooxidation and photoprotection reactions

Photooxidation

6-Thioguanine (21) has a λmax at 340 nm and was found to act as a Type-II photosensitizer, generating singlet oxygen upon UVA irradiation (15) (Scheme 6). The photooxidization products included guanine sulfenate, guanine sulfinate and guanine sulfonate, wherein the structures of the latter two were established unambiguously. Inclusion of N-acetylcysteine during the photooxidation not only protected 21 against oxidation, it produced an addition product, an amino-purinylthiopropanoic acid derivative (22). The photooxidations of tryptophan methyl ester (23) and tryptophan octyl ester (24) were carried out with perinaphthenone (PN) and RB in 1,2-di-oleoyl-glycero-3-phosphatidylcholine (DOPC) vesicles, where 24 intercalated into the membrane to a greater extent due to higher hydrophobicity (16). Chemical and total quenching of 1O2 were enhanced for 23 and 24 in water compared to DOPC liposomes. Tryptophan residues in proteins (25) reacted with 1O2 to form N-formylkynurenine (NFK 26) and hydroperoxide and hydrotryptophan products (17). Here, an anti-NFK antiserum was used in immunological assays, and its usefulness was reaffirmed for the specific detection of oxidized tryptophan residues in cells. One study dealt with the photodegradation of a pesticide, cyanophos (27). Cyanophos 27 was studied in aqueous solution with solar radiation or UV light in the 254–313 nm range, and in the presence and absence of hydroquinone (18). A photooxidation mechanism was implicated because of an increased degradation quantum yield in O2-saturated than O2-free solutions.

Figure Scheme 6..

Photoprotection

Photoprotection mechanisms are also an area of current interest. Recent developments include porous wool fibers that were shown to have enhanced photostability against “photoyellowing” when TiO2 nanoparticles were incorporated into the material as a UV-blocking agent (19). The wool was treated with citric acid as the cross-linking agent and 1-75 g L−1 TiO2 was incorporated onto the wool surface. The inhibition of wool yellowing and oxygenation was attributed to UV blocking of aromatic absorbers, such as tryptophan. Besides TiO2, flavones were another kind of photoprotective agents that were examined in two papers. (1) The photostability of the flavones quercetin (28), morin (29) and rutin (30) was examined with 450 nm light in riboflavin-sensitized photooxidations (Scheme 7) (20). Quercetin 28 was highly susceptible to photooxidation, whereas morin 29 and rutin 30 were less reactive. (2) Due to its photosensitivity, quercetin 28 was shown to stabilize UVA-absorber butyl methoxydibenzoylmethane and UVB-absorber octyl methoxycinnamate compounds (21). Photodegradation studies were followed in oil-in-water emulsions with a Xenon lamp. In this case, quercetin 28 was more effective than the other additives, octocrylene and vitamin E (butylated hydroxyanisole) and functioned to scavenge radicals and chelate metals, thereby slowing lipid peroxidation.

Figure Scheme 7..

Photodynamic action

Major goals in photobiology are to understand how microbes are photoinactivated and tumors are destroyed. MB and RB sensitizers were used to photodynamically inactivate Enterococcus faecalis (22). The E. faecalis cells were examined as suspensions and biofilms. The biofilms were more resistant to photodynamic inactivation compared to the suspensions. Furthermore, it was advantageous to use MB in combination with verapamil hydrochloride (a specific microbial efflux pump inhibitor) to enhance the photodynamic inactivation of the E. faecalis biofilms. As would be expected, the presence of antioxidant pigments such as carotenoids or organic matter can reduce the efficiency of photodynamic inactivation of fungi (23).

Device development played a role in the construction of a hollow-core fiber-optic with an “internal” supply of light and flowing oxygen, and a porous photosensitizer end-capped configuration (24). The device delivered singlet oxygen through a maneuverable fiber tip, with handheld guidance. This led to complete E. coli inactivation when the sensitizing probe tip was immersed in 0.1 mL aqueous samples of 0.1–4.4 × 107 cells over 2 h.

A study of the natural product pyocyanin (1-hydroxy-5-methylphenazine, 31) from the human pathogen bacterium Pseudomonas aeruginosa, and related phenazines (1-methoxy-5-methylphenazine methosulfate [32], 1-hydroxy-phenazine [33] and 5-methylphenazine methosulfate [34]) revealed how structural modifications played a role in the 1O2 quenching (Scheme 8) (25). The total rate constants for 1O2 quenching by pyocyanin 31 and phenazine 33 were rapid in D2O buffer (pD ca 7.2) 4.8 × 108 and 6.8 × 108 m−1 s−1, respectively. Phenazines 32 and 34 were sluggish in quenching 1O2.

Figure Scheme 8..

The use of metal-oxide and polymer-bound photosensitizers has been popular. Photocatalytic bactericidal activity toward E. coli with 30 nm diameter anatase TiO2 nanocrystals was compared with TiO2 powder (Degussa P-25) containing anatase and rutile phases in a ratio of about 3:1 with 400 nm light (26). The former was more active than the latter, which was attributed to less efficient charge recombination and higher ROS yields. Anatase Zr-doped TiO2 nanocrystals (sized ca 25 nm) had a redshifted absorption spectrum (lower bandgap) compared to unmodified TiO2 nanocrystals (27). In the presence of solar radiation, the photoinactivation of P. aeruginosa was enhanced with the Zr-doped TiO2 by about two-fold because the doped samples were less apt to recombine charge. Anatase TiO2 sol particles (spindle shaped, sized ca 50 nm) were synthesized and were found to inactivate H9N2 avian influenza virus (AIV) (28). 365 nm light was used with adjustments to the intensity, irradiation time and quantity of H9N2 AIV, where the inactivation of the AIV viruses reached quantitative levels, 100%. Due to aggregation in aqueous solution, water-dispersed TiO2/PEG nanoparticles were also developed as a photosensitizer system (29). The nanoparticles were examined with monolayer and spheroid C6 rat glioma cells where the cell growth was reduced due to photoexcited TiO2 itself and/or ROS. The TiO2/PEG particles proved to have antitumor activity against C6 monolayer cells, but less so for the C6 spheroids, possibly due to reduced light penetration and low oxygen concentrations in the center of the spheroids.

With the aim of ultimately developing better food storage materials, anthraquinone was covalently incorporated into a polymer (35) (Scheme 9) (30). In the presence of oxygen and UVA radiation, exogenously produced singlet oxygen inactivated microbes that were inoculated onto the polymer surface. Out of the species inoculated (E. coli, Bacillus cereus, Fusarium oxysporum and Saccharomyces cerevisiae), E. coli was the least resistant and B. cereus the most resistant. The biocidal activity involved ROS contact at the microorganism/sensitizing polymer interfaces.

Figure Scheme 9..

Photosensitizer dosimetry was assessed by photoacoustic signals from the photosensitizer in tissue in a study aimed at combating antibiotic-resistant bacteria. In vivo photoacoustic signals were resolved from MB or Photofrin that was injected into burned skin of rats for profiling of the sensitizer up to 3 mm in depth (31). Other themes in photodynamic action were also represented. One technique, chemiluminescent photodynamic antimicrobial therapy (CPAT) used light captured from a chemiluminescent luminol to excite MB or toluidine blue and was effective in killing S. aureus and inhibiting the growth of E. coli (32). In principle, CPAT can bypass the need of an external light source to activate the photosensitizer. Two papers analyzed hematoporphyrins: Hematoporphyrin monomethyl ether (HMME) was used to study PDT effects on choroidal neovascularization (CNV) in rats (33). The photodynamic treatment was performed for 20 min after HMME bolus injection and monitored by fluorescence microscopy where CNV occlusion was observed. Lastly, the photodynamic effects were examined on immature stages of diptera Ceratitis capitata flies with hematoporphyrin IX as the sensitizer (34). The lethal concentration (LC50) of hematoporphyrin IX was found to be 0.173 mm based on a postembryonic development of the insect. Lipid peroxidation was particularly prevalent in the brain and gut of the larvae (34).

Author biographies

Bonnie Kruft was born near Chesapeake Bay in Maryland. She completed her B.S. degree in chemistry at The College of Charleston in Charleston, South Carolina in 2007. She is currently working toward her Ph.D. degree with Professor A. Greer at Brooklyn College of the City University of New York, engaged in computations and spectroscopy of self-replicating and helical molecules. She enjoys activities such as cooking, traveling and photography.

  • image

[ BonnieKruft ]

Alec Greer obtained his Ph.D. degree from the University of Wyoming under Edward L. Clennan. He was a postdoctoral fellow at the University of California, Los Angeles with Christopher S. Foote and then moved to Brooklyn College of the City University of New York in 1999. His research interest is in physical organic chemistry, and is developing devices for use in water disinfection, and as a photochemical surgical knife or singlet oxygen scalpel.

  • image

[ Alec Greer ]

Acknowledgement— We acknowledge support from the National Institute of General Medical Sciences (NIH SC1GM093830).

Ancillary