Mitochondria-targeted cationic porphyrin-triphenylamine hybrids for enhanced two-photon photodynamic therapy

The proof of concept for two-photon activated photodynamic therapy has already been achieved for cancer treatment but the efficiency of this approach still heavily relies on the availability of photosensitizers combining high two-photon absorption and biocompatibility. In this line we recently reported on a series of porphyrin-triphenylamine hybrids which exhibit high singlet oxygen production quantum yield as well as high two-photon absorption cross-sections but with a very poor cellular internalization. We present herein new photosensitizers of the same porphyrin-triphenylamine hybrid series but bearing cationic charges which led to strongly enhanced water solubility and thus cellular penetration. In addition the new compounds have been found localized in mitochondria that are preferential target organelles for photodynamic therapy. Altogether the strongly improved properties of the new series combined with their specific mitochondrial localization lead to a significantly enhanced two-photon activated photodynamic therapy efficiency.     

Light-activated cytotoxic compounds from Malaysian microorganisms for photodynamic therapy of cancer

Photodynamic therapy (PDT) is a promising cancer treatment which involves activation of a photosensitizing drug with light to produce reactive oxygen species that kill tumors without causing damage to unirradiated normal tissues. To date, only Photofrin^®, Foscan^® and Levulan^® have been approved for clinical treatment of cancer. Tropical habitats such as those found in Malaysia are attractive sources of new therapeutic compounds as tremendous chemical diversity is found in a large number of plants, animals, marine- and micro-organisms. In our screening program for novel photosensitizers from nature, colorful strains of fungi (from Aspergillus and Penicillium genus) and bacteria (including actinomycetes and photosynthetic bacteria) were collected from various habitats in Peninsular Malaysia, such as coastal soil, peat soil, marine sponges and wastewater ponds. Methanolic extracts from a total of 85 different species were evaluated with a short-term cell viability assay for photo-cytotoxicity, where a promyelocytic leukemia cell-line, HL60 incubated with 20 μg/ml of extracts was irradiated with 9.6 J/cm² of a broad spectrum light. Two of these extracts, one from Rhodobacter sphaeroides (PBUM003) and one from Rhodopseudomonas palustris (PBUM001) showed moderate to strong photo-cytotoxicity. Subsequent bioassay guided isolation of the PBUM001 extract yielded known photosensitisers that are based on bacteriochlorophyll-a by comparing their molecular weight data, HPLC profiles and UV–vis absorption spectra with literature values, thereby demonstrating the validity of our screening approach.     

Systemic antitumor protection by vascular-targeted photodynamic therapy involves cellular and humoral immunity

Vascular-targeted photodynamic therapy (VTP) takes advantage of intravascular excitation of a photosensitizer (PS) to produce cytotoxic reactive oxygen species (ROS). These ROS are potent mediators of vascular damage inducing rapid local thrombus formation, vascular occlusion, and tissue hypoxia. This light-controlled process is used for the eradication of solid tumors with Pd-bacteriochlorophyll derivatives (Bchl) as PS. Unlike classical photodynamic therapy (PDT), cancer cells are not the primary target for VTP but instead are destroyed by treatment-induced oxygen deprivation. VTP initiates acute local inflammation inside the illuminated area accompanied by massive tumor tissue death. Consequently, in the present study, we addressed the possibility of immune response induction by the treatment that may be considered as an integral part of the mechanism of VTP-mediated tumor eradication. The effect of VTP on the host immune system was investigated using WST11, which is now in phase II clinical trials for age-related macular degeneration and intended to be evaluated for cancer therapy. We found that a functional immune system is essential for successful VTP. Long-lasting systemic antitumor immunity was induced by VTP involving both cellular and humoral components. The antitumor effect was cross-protective against mismatched tumors, suggesting VTP-mediated production of overlapping tumor antigens, possibly from endothelial origin. Based on our findings we suggest that local VTP might be utilized in combination with other anticancer therapies (e.g., immunotherapy) for the enhancement of host antitumor immunity in the treatment of both local and disseminated disease. Y.S. and A.S are the incumbents of the Tillie and Charles Lubin Professorial Chair in Biochemical Endocrinology, and the Robert and Yaddele Sklare Professorial Chair in Biochemistry, respectively. S.J. is the incumbent of the Pauline Recanati Career Development Chair. D.P. in partial fulfillment of her PhD Thesis requirements at the Feinberg graduate school of the Weizmann Institute of Science.     

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.     

Protein microarray for complex apoptosis monitoring of dysplastic oral keratinocytes in experimental photodynamic therapy

Background

Photodynamic therapy is an alternative treatment of muco-cutaneous tumors that uses a light source able to photoactivate a chemical compound that acts as a photosensitizer. The phthalocyanines append to a wide chemical class that encompasses a large range of compounds; out of them aluminium-substituted disulphonated phthalocyanine possesses a good photosensitizing potential.

Results

The destructive effects of PDT with aluminium-substituted disulphonated phthalocyanine are achieved by induction of apoptosis in tumoral cells as assessed by flow cytometry analysis. Using protein microarray we evaluate the possible molecular pathways by which photodynamic therapy activates apoptosis in dysplastic oral keratinocytes cells, leading to the tumoral cells destruction. Among assessed analytes, Bcl-2, P70S6K kinase, Raf-1 and Bad proteins represent the apoptosis related biomolecules that showed expression variations with the greatest amplitude.

Conclusions

Up to date, the intimate molecular apoptotic mechanisms activated by photodynamic therapy with this type of phthalocyanine in dysplastic human oral keratinocytes are not completely elucidated. With protein microarray as high-throughput proteomic approach a better understanding of the manner in which photodynamic therapy leads to tumoral cell destruction can be obtained, by depicting apoptotic molecules that can be potentially triggered in future anti-tumoral therapies.     

Aminosquaraines as potential photodynamic agents: Synthesis and evaluation of in vitro cytotoxicity

The synthesis of several aminosquaraine cationic dyes displaying strong absorption within the so-called phototherapeutic window (650–850 nm) is described. Their cytotoxicity, under dark and illuminated conditions, was tested against several human tumor cell lines (breast, lung, cervical and hepatocellular carcinomas) and non-tumor porcine liver primary cells. All compounds showed to inhibit the growth of the tumor cells upon irradiation more than in the absence of light, in more or less extension, clearly exhibiting photodynamic activity. The photosensitizing ability against some cell lines, together with the low toxicity for the non-tumor primary PLP2 cells displayed by some of the compounds synthetized, turns them into potential candidates as photosensitizers for PDT.     

光动力疗法治疗感染性疾病的动物模型

光动力疗法(photodynamic therapy,PDT)是利用特定波长的激发光照射生物靶标上的光敏剂,从而产生活性氧并有效杀伤多种耐药病原体的新型治疗方式,具有作用广、安全可控、不易耐受等优点。大量体外实验已证实了PDT疗效,但目前动物实验数据较少,且治疗参数不一,一定程度上影响了PDT在临床治疗中的广泛应用。本文综述近年来PDT用于体内抗感染治疗的动物模型构建、治疗方案设计等方面的研究进展,为未来PDT抗感染研究及临床应用提供参考。     

Effect of intermittency factor on singlet oxygen and PGE2 formation in azulene-mediated photodynamic therapy: A preliminary study

In photodynamic therapy, intermittent irradiation modes that incorporate an interval between pulses are believed to decrease the effect of hypoxia by permitting an interval of re-oxygenation. The effect of the irradiation intermittency factor (the ratio of the irradiation pulse time to the total irradiation time) on singlet oxygen formation and inflammatory cytokine production was examined using azulene as a photosensitizer. Effects of difference intermittency factor on singlet oxygen formation and inflammatory cytokine were examined. Azulene solutions (1/10 μM) were irradiated with a 638-nm 500 mW diode laser in fractionation (intermittency factor of 5 or 9) or continuous mode using 50 mW/cm² at 4 or 8 J/cm². Singlet oxygen measurement was performed using a dimethyl anthracene probe. Peripheral blood mononuclear cells (PBMC) were stimulated by 10 ng/ml rhTNF-α for 6 h, before addition of 1 and 10 μM azulene solutions and irradiation. PGE₂ measurement was undertaken using a human PGE₂ ELISA kit. Kruskal-Wallis with Dunn Bonferroni test was used for statistical analyses at p < 0.05.Irradiation of 1 μM azulene+4 J/cm²+intermittency factor of 9 increased singlet oxygen 3-fold (p < 0.0001). Irradiation of 10 μM azulene at either 4 J/cm²+intermittency of 9 or 8 J/cm²+intermittency factor of 5 reduced PGE₂ expression in PBMCs to non-inflamed levels. Thus, at 50 mW/cm², 10 μM azulene-mediated photodynamic therapy with a high intermittency factor and a low energy density generated sufficient singlet oxygen to suppress PGE₂ in Inflamed PBMCs.     

Histomorphological changes in murine fibrosarcoma after hypericin-based photodynamic therapy

Histomorphological changes in murine fibrosarcoma after photodynamic therapy (PDT) based on the natural photosensitizer hypericin were evaluated. C3H/DiSn mice were inoculated with fibrosarcoma G5:1:13 cells. When the tumour reached a volume of 40-80 mm(3) the mice were intraperitoneally injected with hypericin, either in a single dose (5 mg/kg; 1 or 6 h before laser irradiation) or two fractionated doses (2.5 mg/kg; 6 and 1 h before irradiation with laser light; 532 nm, 70 mW/cm(2), 168 J/cm(2)). All groups of PDT-treated animals with single and fractionated hypericin dosing presented primary vascular reactions including vascular dilatation, congestion, thrombosis and oedema. Two hours after PDT there were necrotic changes with small, rather focal appearance. One day after therapy the necrotic areas were enhanced, often affecting a complete superficial layer of tumour tissue. Necrotic areas were accompanied with inflammation and haemorrhages.     

Extended rhodamine photosensitizers for photodynamic therapy of cancer cells

Extended thio- and selenorhodamines with a linear or angular fused benzo group were prepared. The absorption maxima for these compounds fell between 640 and 700 nm. The extended rhodamines were evaluated for their potential as photosensitizers for photodynamic therapy in Colo-26 cells. These compounds were examined for their photophysical properties (absorption, fluorescence, and ability to generate singlet oxygen), for their dark and phototoxicity toward Colo-26 cells, and for their co-localization with mitochondrial-specific agents in Colo-26 and HUT-78 cells. The angular extended rhodamines were effective photosensitizers toward Colo-26 cells with 1.0 J cm⁻² laser light delivered at λ_max ± 2 nm with values of EC₅₀ of (2.8 ± 0.4) × 10⁻⁷ M for sulfur-containing analogue 6-S and (6.4 ± 0.4) × 10⁻⁸ M for selenium-containing analogue 6-Se. The linear extended rhodamines were effective photosensitizers toward Colo-26 cells with 5 and 10 J cm⁻² of broad-band light (EC₅₀’s ⩽ 2.4 × 10⁻⁷ M).     

Dynamic model of vascular-targeted photodynamic therapy

Vascular-targeted photodynamic therapy has shown efficiency in treating port wine stains. A dynamic model that incorporates blood flow, kinetic diffusion, oxygen and photosensitizer consumption and reaction, and light modulation is proposed to reveal the interactions among light, photosensitizer, and oxygen. Simulation results show that pulse light modulation synchronized with heartbeats hold the advantage of increased singlet oxygen accumulation, higher oxygen concentration and lower temperature. Meanwhile, constant light treatment is advantageous in terms of higher temperature, lower total oxygen concentration and singlet oxygen accumulation. Therefore, the optimized treatment protocol may involve a balance among the phototoxicity, hypoxia, and photothermolysis.     

System identification of photosensitiser uptake kinetics in photodynamic therapy

This study draws the interest of system identification to the experimental modelling of in vitro uptake kinetics of photosensitising agents (PS) into cancer cells. The proposed identification methodology must be usable and valid for every PS. Therefore, three PSs characterized by opposing chemical and biological properties have been selected: (1) PC: a second generation photosensitising agent conjugated via a spacer to a VEGF receptor-specific heptapeptide, (2) Ce6: Chlorin e6, and (3) TPP: TetraPhenylPorphyrin. Experiments have been carried out with two rates (2% and 9%) of foetal bovin serum in the culture medium and one cancer cell line U87 a human malignant glioma. Difficulties of such an application are triple: (i) lack of data, (ii) low signal-to-noise ratio and (iii) ‘poor’ stimulus signals. The proposed identification methodology deals with the design of experiments, the selection of a model structure, the estimation of the model parameters and the estimation of the parameter uncertainties. The photosensitiser uptake phenomenon is described by a first-order transfer function. Estimates of the time constant and the static gain provide quantitative information about the uptake rate and yield of the PS. The parameter uncertainty is described by confidence regions in parameters space. This representation is presented as an efficient way to discriminate the uptake characteristics of different photosensitisers. This representation also emphasizes the effects of some biological factors, such as the serum rate, on the uptake yield.     

Tumor microenvironment-activated nanosystems with selenophenol substituted BODIPYs as photosensitizers for photodynamic therapy

NIR-light-absorbing photosensitizers with the capability of selective localization and activation in tumor regions are of great importance for practical photodynamic therapy (PDT). Here, selenophenol substituted BODIPYs were designed and synthesized as new photosensitizers for PDT. One of these obtained BODIPYs, IBSeOV, possesses an intense and low energy absorption with a high singlet oxygen quantum yield (Φ_Δ = 60%). Considering manganese dioxide (MnO₂) nanosheets as versatile nanocarriers in cancer theranostics, nanosystem IBSeOV/MnO₂ was then fabricated to furnish tumor environment selective activation. Such designed nanoplatform allowed for GSH-controllable ¹O₂ production and exhibited low cytotoxicity in dark but good photocytotoxicity to cancer cells. The in vivo antitumor outcome suggested the high treatment efficiency of IBSeOV/MnO₂ for tumor therapy.     

Mitochondria are targets of photodynamic therapy

Photodynamic Therapy (PDT) is an evolving cancer treatment that depends on three known and variable components: photosensitizer, light and oxygen. Optimization of these variables yields reactive oxygen species, mainly singlet oxygen, that damage cellular components leading to cytotoxicity. Our research has demonstrated that porphyrin sensitizers, in particular, significantly inhibit the inner mitochondrial membrane enzymes cytochrome c oxidase and F ₀ F ₁ ATP synthase. These results were obtained from an in vivo-in vitro experimental protocol that exposes sensitizers to metabolic and pharmacokinetic events. The resulting inhibition of oxidative phosphorylation was expected to reduce ATP levels, which were quantitated in cells and were confirmed by ³¹P-NMR spectroscopy of tumors in situ in animals treated with PDT. Based on these findings, and more recent investigations of apoptosis, there is little doubt that mitochondria are critical targets in the actions of PDT.     

Online electrochemical monitoring of nitric oxide during photodynamic therapy

Photodynamic therapy (PDT), as a novel treatment modality, is based on the use of a photosensitizing agent with an excitation light source for the treatment of various malignancies. Its effect is mediated through reactive oxygen species and nitric oxide (NO), which are shown to be present in apoptosis. Individual differences among patients and even in different areas of the same tumor in one patient may cause a major problem with PDT: dose calculation during application of the light. An electrochemical sensor is proposed for online monitoring of NO generation as a solution of this problem. 5-Aminolevulinic acid (ALA) was administered as the photosensitizer in rat cerebellum. An amperometric sensor, selective to NO, was designed and tested both in vitro and in vivo during PDT. ALA-mediated PDT resulted in rapid generation of NO, starting as early as the application of light on the tissue. Simultaneous amperometric recordings have been carried out for 5 min during PDT. The progressive increase in NO concentration peaked at 1.10 min and then the response current began to decrease until it reached a plateau at around 70% of its peak value. This study, for the first time, electrochemically demonstrates the generation of NO during PDT. Rapid and stable responses obtained by the experimental setup confirmed that this method could be used as an online monitoring system for PDT-mediated apoptosis.     

5-氨基酮戊酸光动力疗法对不同疾病来源的耐甲氧西林金黄色葡萄球菌的体外杀伤作用

光动力疗法(photodynamic therapy,PDT)利用光敏剂与光源反应后产生的活性氧,破坏细菌组分,进而致细菌死亡。其多靶位杀伤特性在治疗耐甲氧西林金黄色葡萄球菌(methicillin-resistant Staphylococcus aureus,MRSA)感染方面有应用前景,但相关研究尚处于起步阶段。本研究MRSA菌株取自烧伤、急性咽炎、鼻窦炎和肺炎4类临床常见MRSA感染性疾病患者,使用5-氨基酮戊酸(5-aminolevulinic acid,ALA)光敏剂、发光二极管光源,于体外检测ALA介导的PDT(ALA-PDT)对MRSA菌株的杀伤作用。结果显示,经5mmol/L ALA孵育1h后,给予360J/cm~2强光[(633±10)nm]照射1h,ALA-PDT对MRSA菌株具有1.80log_(10)cfu的有效杀伤作用。结果提示,在相同实验条件和参数下,ALA-PDT对上述4种疾病来源的MRSA菌株体外杀伤作用无统计学差异。     

Synthesis and biological evaluation of new boron-containing chlorin derivatives as agents for both photodynamic therapy and boron neutron capture therapy of cancer

New boron-containing chlorin derivatives 9 and 13 as agents for both photodynamic therapy (PDT) and boron neutron capture therapy (BNCT) of cancer were synthesized from photoprotoporphyrin IX dimethyl ester (2) and l-4-boronophenylalanine-related compounds. The in vivo biodistribution and clearance of 9 and 13 were investigated in tumor-bearing mice. The time to maximum accumulation of compound 13 in tumor tissue was one-fourth of that of compound 9, and compound 13 showed rapid clearance from normal tissues within 24 h after injection. The in vivo therapeutic efficacy of PDT using 13 was evaluated by measuring tumor growth rates in tumor-bearing mice with 660 nm light-emitting diode irradiation at 3 h after injection of 13. Tumor growth was significantly inhibited by PDT using 13. These results suggested that 13 might be a good candidate for both PDT and BNCT of cancer.     

A non-aggregated silicon(IV) phthalocyanine-lactose conjugate for photodynamic therapy

To develop a highly efficient photosensitizer for photodynamic therapy (PDT), we have designed and synthesized a phthalocyanine-lactose conjugate (Pc-Lac) through axial modification of silicon(IV) phthalocyanine with lactose moieties. With the lactose substituents, Pc-Lac is highly hydrophilic and non-aggregated with efficient reactive oxygen species (ROS) generation in aqueous media. With these desirable properties, Pc-Lac shows high photocytotoxicity and cellular uptake toward HepG2 cells. In addition, in vivo fluorescence imaging shows that Pc-Lac could selectively remain at tumor site, leading to its enhanced photodynamic efficacy against H22 tumor-bearing mice. Therefore, Pc-Lac shows a great potential as a highly efficient molecular photosensitizer for PDT.     

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。