A naphthalocyanine based near-infrared photosensitizer: Synthesis and in vitro photodynamic activities

A hydrophilic near-infrared (NIR) photosensitizer featuring a naphthalocyanine core and peripheral carboxylate acid groups was synthesized and characterized. Its photophysical and photochemical properties were studied and compared with phthalocyanine. Due to the extended π-conjugation, both the Q band and fluorescence emit of this naphthalocyanine bathochromically shift to NIR region. It also exhibits superior NIR photodynamic efficiency to phthalocyanine as evidenced by high efficiency in generating singlet oxygen (Φ_Δ = 0.66) and in vitro phototoxicity toward Hela human cervical cancer cells. Therefore, this novel naphthalocyanine could potentially be a NIR photosensitizer for photodynamic therapy.     

A molecular hybrid producing simultaneously singlet oxygen and nitric oxide by single photon excitation with green light

Combination of photosensitizers (PS) for photodynamic therapy with NO photodonors (NOPD) is opening intriguing horizons towards new and still underexplored multimodal anticancer and antibacterial treatments not based on “conventional” drugs and entirely controlled by light stimuli. In this contribution, we report an intriguing molecular hybrid based on a BODIPY light-harvesting antenna that acts simultaneously as PS and NOPD upon single photon excitation with the highly biocompatible green light. The presented hybrid offers a combination of superior advantages with respect to the other rare cases reported to date, meeting most of the key criteria for both PSs and NOPDs in the same molecular entity such as: (i) capability to generate ¹O₂ and NO with single photon excitation of biocompatible visible light, (ii) excellent ¹O₂ quantum yield and NO quantum efficiency, (iii) photogeneration of NO independent from the presence of oxygen, (iv) large light harvesting properties in the green region. Furthermore, this compound together with its stable photoproduct, is well tolerated by both normal and cancer cells in the dark and exhibits bimodal photomortality of cancer cells under green light excitation due to the combined action of the cytotoxic ¹O₂ and NO.     

Folic acid conjugates with photosensitizers for cancer targeting in photodynamic therapy: Synthesis and photophysical properties

Recent researches in photodynamic therapy have focused on novel techniques to enhance tumour targeting of anticancer drugs and photosensitizers. Coupling a photosensitizer with folic acid could allow more effective targeting of folate receptors which are over-expressed on the surface of many tumour cells. In this study, different folic acid–OEG-conjugated photosensitizers were synthesized, characterized and their photophysical properties were evaluated. The introduction of an OEG does not significantly improve the hydrophilicity of the FA–porphyrin. All the FA-targeted photosensitizers present good to very good photophysical properties. The best one appears to be Ce6. Molar extinction coefficient, fluorescence and singlet oxygen quantum yields were determined and were compared to the corresponding photosensitizer alone.     

Kinetics of singlet oxygen photosensitization in human skin fibroblasts

The roles played by singlet oxygen (¹O₂) in photodynamic therapy are not fully understood yet. In particular, the mobility of ¹O₂ within cells has been a subject of debate for the last two decades. In this work, we report on the kinetics of ¹O₂ formation, diffusion, and decay in human skin fibroblasts. ¹O₂ has been photosensitized by two water-soluble porphyrins targeting different subcellular organelles, namely the nucleus and lysosomes, respectively. By recording the time-resolved near-IR phosphorescence of ¹O₂ and that of its precursor the photosensitizer's triplet state, we find that the kinetics of singlet oxygen formation and decay are strongly dependent on the site of generation. ¹O₂ photosensitized in the nucleus is able to escape out of the cells while ¹O₂ photosensitized in the lysosomes is not. Despite showing a lifetime in the microsecond time domain, ¹O₂ decay is largely governed by interactions with the biomolecules within the organelle where it is produced. This observation may reconcile earlier views that singlet oxygen-induced photodamage is highly localized, while its lifetime is long enough to diffuse over long distances within the cells.     

Synthesis,spectroscopic studies and biological evaluation of acridine derivatives: The role of aggregation on the photodynamic efficiency

Two new photoactive compounds (1 and 2) derived from the 9-amidoacridine chromophore have been synthesized and fully characterized. Their abilities to produce singlet oxygen upon irradiation have been compared. The synthesized compounds show very different self-aggregating properties since only 1 present a strong tendency to aggregate in water. Biological assays were conducted with two cell types: hepatoma cells (Hep3B) and human umbilical vein endothelial cells (HUVEC). Photodynamic therapy (PDT) studies carried out with Hep3B cells showed that non-aggregating compound 2 showed photoxicity, ascribed to the production of singlet oxygen, being aggregating compound 1 photochemically inactive. On the other hand suspensions of 1, characterized as nano-sized aggregates, have notable antiproliferative activity towards this cell line in the dark.     

A comparative study of fluorescent singlet oxygen probes in plant leaves

Four fluorescent singlet oxygen sensors: DanePy, its oxalate salt, Singlet Oxygen Sensor Green and MVP, were infiltrated into tobacco leaves and tested for toxicity, subcellular localization, light sensitivity and capacity to trap the singlet oxygen produced in photoinhibition. For reference, a broad sensitivity free radical probe, TEMPO-9-AC, was also included. Photochemical yield was approximately 15% and 10% inhibited by Singlet Oxygen Sensor Green and MVP, respectively, but was not significantly affected by the other probes. Under photoinhibitory conditions, brought about by irradiating lincomycin-treated leaves with strong photosynthetically active radiation, DanePy and Singlet Oxygen Sensor Green were responsive. Singlet Oxygen Sensor Green was also reactive to low, non-photoinhibitory light exposure of the leaf, which was not characteristic to the other probes. MVP did not respond to singlet oxygen which can partly be explained by a possible attenuation of its blue emission in the leaf, as shown by the example TEMPO-9-AC. DanePy-oxalate did not respond to photosynthetic singlet oxygen due to lack of its penetration into photosynthetic tissue and hence could be useful in detecting any singlet oxygen which escapes from a chloroplast initiation site. DanePy was localized in the chloroplasts, while Singlet Oxygen Sensor Green was mainly found in the epidermal cells preferentially associated with the nucleus.      

High optical-throughput spectroscopic singlet oxygen and photosensitizer luminescence dosimeter for monitoring of photodynamic therapy

We report a high light-throughput spectroscopic dosimeter system that is able to noninvasively measure luminescence signals of singlet oxygen (¹O₂) produced during photodynamic therapy (PDT) using a CW (continuous wave) light source. The system is based on a compact, fiber-coupled, high collection efficiency spectrometer (>50% transmittance) designed to maximize optical throughput but with sufficient spectral resolution (~7 nm). This is adequate to detect ¹O₂ phosphorescence in the presence of strong luminescence background in vivo. This system provides simultaneous acquisition of multiple spectral data points, allowing for more accurate determination of luminescence baseline via spectral fitting and thus the extraction of ¹O₂ phosphorescence signal based solely on spectroscopic decomposition, without the need for time-gating. Simultaneous collection of photons at different wavelengths improves the quantum efficiency of the system when compared to sequential spectral measurements such as filter-wheel or tunable-filter based systems. A prototype system was tested during in vivo PDT tumor regression experiments using benzoporphyrin derivative (BPD) photosensitizer. It was found that the treatment efficacy (tumor growth inhibition rate) correlated more strongly with ¹O₂ phosphorescence than with PS fluorescence. These results indicate that this high photon-collection efficiency spectrometer instrument may offer a viable option for real-time ¹O₂ dosimetry during PDT treatment using CW light.     

The effect of TSPP-mediated photodynamic therapy and Parecoxib in experimental tumours

Aims

The study investigated the effects of the combined treatment Parecoxib (Pcox) and 5,10,15,20-tetra-sulphonato-phenyl-porphyrin(TSPP)-mediated photodynamic therapy on Walker 256 carcinosarcoma.

Main methods

Five groups of male Wistar rats were used: the control group, treated with TSPP, group 2, irradiated 24 h thereafter, group 3, treated with Pcox and irradiated 24 h thereafter, groups 4 and 5 treated with combined therapies, TSPP and Pcox before irradiation, and Pcox 24 h after TSPP and irradiation respectively. Tumour inflammation, growth and non-growth factors, apoptosis/necrosis rate and oxidative/nitrosative stress markers were investigated.

Key findings

Malondialdehyde levels and cyclooxygenase (COX)-2 expression increased significantly in the group treated with Pcox after TSPP-PDT when compared with TSPP + IR group (p < 0.05, p < 0.001 respectively), in correlation with a decrease in glutathione levels (p < 0.05). The quantification of apoptosis, based on the TUNEL-assay, and necrosis rate revealed an increase of apoptotic/necrotic index in the same group (p < 0.05). On the other hand, Pcox administered before irradiation showed a significant increase in both vascular endothelial growth factor (VEGF) and COX-2 levels (p < 0.05) and in nitric oxide production (p < 0.01), when compared with the control group.

Significance

The administration of Pcox after TSPP-mediated PDT showed promising antitumoural effects, leading to an increase in oxidative and nitrosative stress as well as apoptosis/necrosis rate in tumour tissue. These results show that combined regimens that involve selective COX-2 inhibitors administration after irradiation may improve the therapeutic effectiveness of PDT.     

Influence of the pH on the photodynamic effect in lysozyme A comparative kinetic study with the sensitized photooxidation of isolated amino acids

Summary The kinetics of the eosin-sensitized photooxidation ([O₂(¹_g)]-mediated) of the protein lysozyme (Lyso) was investigated under two different pH conditions (pH 7 and pH 11). Rates of oxygen consumption and the fade in the protein fluorescence spectrum upon sensitized irradiation were monitored. Parallel studies on both denatured Lyso (absence of the four-S-S- bridges in the protein) and different mixtures of the photooxidizable amino acids of Lyso were also carried out. The mixtures maintained the same molar ratio as in the native protein, and were selected just in order to throw into relief the preferential amino acids that were being photooxidized at both pH values.Under work conditions Lyso was only photooxidizable at pH 7, whereas the opposite accounted for the denatured protein: only measurable oxygen consumption was detected at pH 11. Nevertheless, Lyso at pH 11, evidenced an important physical quenching of O₂(¹_g) due to the Tyr and Trp residues.The results for the native protein were interpreted on the basis of a previously described dark complex Eosin-Lyso, which selectively favours the photooxidation of the bounded protein. The Trp residues were the main reactive entities in the native protein. The photodinamic effect in denatured Lyso was characterized by the prevalence of Tyr residues as photooxidizable targets.In the discussion of the results, a comparisson with the photooxidation kinetics of the mixtures of free amino acids was made.Abbreviations O₂(³_g ^–) ground state triplet oxygen - O₂(¹_g) singlet molecular oxygen - Lyso lysozyme - LysoD denatured lysozyme - Eos eosin - FFA furfuryl alcohol - Trp tryptophan - Tyr tyrosine - Cys cysteine - Cis cystine - Met methionine - His histidine - AA amino acid - a.u. arbitrary units     

Production of singlet oxygen on irradiation of a photodynamic therapy agent, zinc-coproporphyrin III, with low host toxicity

Zinc-coproporphyrin III (Zincphyrin) acts efficiently as a photodynamic therapy (PDT) agent in mice, while it shows no tumor cell-killing activity in vitro and has a high LD₅₀ (low toxicity) in mice. It appears to have advantages over other porphyrins as a practical PDT reagent. In order to examine the action mechanism of Zincphyrin in PDT, we evaluated the photochemical characteristics of Zincphyrin by measurement of the near-infrared emission at 1268 nm, which provides direct evidence for formation of ¹O₂. Intense emission was observed in the presence of Zincphyrin, and was completely inhibited by NaN₃, a ¹O₂ scavenger. Based on a quenching study, the rate constant of the reaction of ¹O₂ with NaN₃ was determined to be 1.5–3.5 M^–1 s^–1, which is close to the reported value (3.8×10⁸ M^–1 s^–1). The intensity of the ¹O₂-specific emission was proportional to both the laser power and the concentration of Zincphyrin. The fluorescence quantum yield of Zincphyrin was 0.004 in phosphate buffer (100 mM, pH 7.4), which indicates that the excited state decays via other pathway(s) faster than through the fluorescence emission pathway. The lifetime of the triplet state of Zincphyrin (210 s) was relatively long compared to that of other porphyrins, such as hematoporphyrin (Hp) (40 s), coproporphyrin I (50 s), or coproporphyrin III (36 s). These results demonstrate the photodynamic generation of ¹O₂ by Zincphyrin.     

The role of strong hypoxia in tumors after treatment in the outcome of bacteriochlorin-based photodynamic therapy

Blood flow and pO₂ changes after vascular-targeted photodynamic therapy (V-PDT) or cellular-targeted PDT (C-PDT) using 5,10,15,20-tetrakis(2,6-difluoro-3-N-methylsulfamoylphenyl) bacteriochlorin (F₂BMet) as photosensitizer were investigated in DBA/2 mice with S91 Cloudman mouse melanoma, and correlated with long-term tumor responses. F₂BMet generates both singlet oxygen and hydroxyl radicals under near-infrared radiation, which consume oxygen. Partial oxygen pressure was lowered in PDT-treated tumors and this was ascribed both to oxygen consumption during PDT and to fluctuations in oxygen transport after PDT. Similarly, microcirculatory blood flow changed as a result of the disruption of blood vessels by the treatment. A novel noninvasive approach combining electron paramagnetic resonance oximetry and laser Doppler blood perfusion measurements allowed longitudinal monitoring of hypoxia and vascular function changes in the same animals, after PDT. C-PDT induced parallel changes in tumor pO₂ and blood flow, i.e., an initial decrease immediately after treatment, followed by a slow increase. In contrast, V-PDT led to a strong and persistent depletion of pO₂, although the microcirculatory blood flow increased. Strong hypoxia after V-PDT led to a slight increase in VEGF level 24 h after treatment. C-PDT caused a ca. 5-day delay in tumor growth, whereas V-PDT was much more efficient and led to tumor growth inhibition in 90% of animals. The tumors of 44% of mice treated with V-PDT regressed completely and did not reappear for over 1 year. In conclusion, mild and transient hypoxia after C-PDT led to intense pO₂ compensatory effects and modest tumor inhibition, but strong and persistent local hypoxia after V-PDT caused tumor growth inhibition.     

Encapsulation of methylene blue in polyacrylamide nanoparticle platforms protects its photodynamic effectiveness

The ability to prevent methylene blue (MB), a photosensitizer, from being reduced by plasma reductases will greatly improve its efficacy in photodynamic therapy (PDT) applications. We have developed a delivery approach for PDT by encapsulating MB using nanoparticle platforms (NPs). The 30-nm polyacrylamide-based NPs provide protection for the embedded MB against reduction by diaphorase enzymes. Furthermore, our data shows the matrix-protected MB efficiently induces photodynamic damage to tumor cells. The unprecedented results demonstrate the significant in vitro photodynamic effectiveness of MB when encapsulated within NPs, which promises to open new opportunities for MB in its in vivo and clinical studies.     

Two-photon irradiation of an intracellular singlet oxygen photosensitizer: Achieving localized sub-cellular excitation in spatially-resolved experiments

Abstract

The response of a given cell to spatially-resolved sub-cellular irradiation of a singlet oxygen photosensitizer (protoporphyrin IX, PpIX) using a focused laser was assessed. In these experiments, incident light was scattered over a volume greater than that defined by the dimensions of the laser beam as a consequence of the inherent inhomogeneity of the cell. Upon irradiation at a wavelength readily absorbed by PpIX in a one-photon transition, this scattering of light eliminated any advantage accrued to the use of focused irradiation. However, upon irradiation at a longer wavelength where PpIX can only absorb light under non-linear two-photon conditions, meaningful intracellular resolution was achieved in the small spatial domain where the light intensity was high enough for absorption to occur.     

光动力疗法对体外培养人胰腺癌细胞株PANC-1作用

目的:探讨血卟啉注射液(Hematoporphyrin Derivative,HPD)光动力疗法(Photodynamic Therapy,PDT)对体外培养的人胰腺癌细胞株PANC-1的生物作用。方法:实验分为4组,空白对照组、单纯HPD组、单纯光照组及HPD+PDT组。采用MTT法检测光动力作用后细胞的存活率,并用Annexin V-FITC/P I双染法检测其凋亡率。结果:在光敏剂浓度为5mg/L,光照剂量为10J/cm2时,光动力对PANC-1细胞达到最佳的实验效果,与对照组相比差异有显著性。在此实验参数条件下,流式细胞术(FCM)检测各组人胰腺癌细胞PANC-1凋亡率:HPD+PDT实验组达(36.40±4.21)%,明显高于单纯HPD(6.76±0.44)%,单纯PDT组(8.30±0.32)%及空白组(5.00±0.53)%三个对照组(P<0.05),三个对照组间差异无统计学意义(P>0.05)。结论:PDT光动力作用对体外培养人胰腺癌细胞PANC-1有明确抑制效应,并与HPD浓度及光照强度相关。     

Photogeneration of free radicals (*OH and HB*-) and singlet oxygen (1O2) by hypocrellin B in TX-100 micelles microsurroundings

To solve the problems faced in clinical use of hypocrellins, a water-soluble preparation of Hypocrellin B (HB), HB-Triton X-100 (TX-100) micelles, was prepared. To evaluate the photodynamic activity, the free radicals (^•OH and HB^•¯) and singlet oxygen (

1

O

2

) generated via photosensitization of the preparation in aqueous solution were detected by using electron paramagnetic resonance (EPR) and spectrophotometric methods. It was observed that

1

O

2

was formed with a quantum yield of 0.72, similar to that for HB in organic solvents, further, hydroxyl radicals (

•

OH) could also be efficiently produced by the new preparation, which have never before been detected following HB photoactivities. In addition, the semiquinone anion radicals (HB^•-) could also be generated via the self-electron transfer between an excited triplet state and a ground state molecule. The accumulation of HB^•- would replace that of

•

OH or

1

O

2

with the depletion of oxygen in the system. All these findings suggested that the HB-TX-100 micelles could play the photodynamic action through not only the type I mechanism by free radicals (^•OH, O₂^•- and HB^•-) but also the type II mechanism by singlet oxygen (

1

O

2

). It can be concluded further that the new preparation basically maintains the inherent photodynamic activity of HB, or even higher.     

Generation and suppression of singlet oxygen in hair by photosensitization of melanin

We have studied the spectroscopic properties of hair (white, blond, red, brown, and black) under illumination with visible light, giving special emphasis to the photoinduced generation of singlet oxygen (¹O₂). Irradiation of hair shafts (λ_ex > 400 nm) changed their properties by degrading the melanin. Formation of C3 hydroperoxides in the melanin indol groups was proven by ¹H NMR. After 532-nm excitation, all hair shafts presented the characteristic ¹O₂ emission (λ_em = 1270 nm), whose intensity varied inversely with the melanin content. ¹O₂ lifetime was also shown to vary with hair type, being five times shorter in black hair than in blond hair, indicating the role of melanin as a ¹O₂ suppressor. Lifetime ranged from tenths of a nanosecond to a few microseconds, which is much shorter than the lifetime expected for ¹O₂ in the solvents in which the hair shafts were suspended, indicating that ¹O₂ is generated and suppressed inside the hair structure. Both eumelanin and pheomelanin were shown to produce and to suppress ¹O₂, with similar efficiencies. The higher amount of ¹O₂ generated in blond hair and its longer lifetime is compatible with the stronger damage that light exposure causes in blond hair. We propose a model to explain the formation and suppression of ¹O₂ in hair by photosensitization of melanin with visible light and the deleterious effects that an excess of visible light may cause in hair and skin.     

Effect of curcumin-nanoemulsion associated with photodynamic therapy in breast adenocarcinoma cell line

Curcumin, a natural compound has several antineoplastic activities and is a promising natural photosensitizer used in photodynamic therapy. However, its low solubility in physiological medium limit the clinical use of curcumin. This study aimed to analyze the action of curcumin-nanoemulsion, a new and well-designed Drug Delivery System (DDS+) molecule, used as a photosensitizing agent in photodynamic therapy in an in vitro breast cancer model, MCF-7 cells. The empty nanoemulsion fulfils all necessary requirements to be an excellent DDS. Furthermore, the use of curcumin-nanoemulsion in photodynamic therapy resulted in a high phototoxic effect after activation at 440?nm, decreasing to <10% viable tumor cells after two irradiations and increasing the reactive oxygen species (ROS) production. The use of curcumin-nanoemulsion associated with photodynamic therapy resulted in an increase in the levels of caspase 3/7 activity for the studied MCF-7 cell model, indicating that this therapy triggers a cascade of events that lead to cell death, such as cellular apoptosis. In conclusion, curcumin-nanoemulsion proved to be efficient as a photosensitizing agent, had phototoxic effects, significantly decreased the proliferation of MCF-7 cells and stimulating the ROS production in combination with photodynamic therapy, so, this formulation has a great potential for use in treatment of breast cancer.     

Scope and limitations of the TEMPO/EPR method for singlet oxygen detection: the misleading role of electron transfer

For many biological and biomedical studies, it is essential to detect the production of ¹O₂ and quantify its production yield. Among the available methods, detection of the characteristic 1270-nm phosphorescence of singlet oxygen by time-resolved near-infrared (TRNIR) emission constitutes the most direct and unambiguous approach. An alternative indirect method is electron paramagnetic resonance (EPR) in combination with a singlet oxygen probe. This is based on the detection of the TEMPO free radical formed after oxidation of TEMP (2,2,6,6-tetramethylpiperidine) by singlet oxygen. Although the TEMPO/EPR method has been widely employed, it can produce misleading data. This is demonstrated by the present study, in which the quantum yields of singlet oxygen formation obtained by TRNIR emission and by the TEMPO/EPR method are compared for a set of well-known photosensitizers. The results reveal that the TEMPO/EPR method leads to significant overestimation of singlet oxygen yield when the singlet or triplet excited state of the photosensitizer is efficiently quenched by TEMP, acting as electron donor. In such case, generation of the TEMP⁺ radical cation, followed by deprotonation and reaction with molecular oxygen, gives rise to an EPR-detectable TEMPO signal that is not associated with singlet oxygen production. This knowledge is essential for an appropriate and error-free application of the TEMPO/EPR method in chemical, biological, and medical studies.