Photophysical properties and photocytotoxicity of novel phthalocyanines - potentially useful for their application in photodynamic therapy

Two novel zinc(II) phthalocyanines bearing non-peripheral ester-alkyloxy substituents (Pc-4 and Pc-5) were synthesized, by a two-step procedure starting from 2,3-dicyanohydroquinone. Both sensitizers show promising photophysical properties, including solvatochromic study, qualitative evaluation of emission, aggregation behavior and singlet oxygen generation. It was proven that the photodynamic activity of the compounds studied depends on the molecular oxygen level. Comparison of the quantum yields of singlet oxygen generation as well as the oxidation rate constants using 1,3-diphenylisobenzofurane before and after deaeration proved the photodynamic effect of Pc-4 and Pc-5 to be governed by the photosensitization mechanism II. Changes in the quantities of the compounds exposed to irradiation were also estimated. Upon their exposure to light the changes in intensity of the Q band were monitored. The photodecomposition of Pc-4 and Pc-5 in DMSO or DMF solutions was found to proceed according to the first order kinetic reaction in two stages.The photosensitizing effect of Pc-4 in HSC-3 cells was significantly lower than that of ZnPc. Pc-4 was ineffective at 0.1 μM, while a low, approx. 15% photocytotoxicity was observed at 5 μM, at a distance of 5 and 10 cm. The efficacy of Pc-4 in photokilling of cultured cells is affected by hydrophobicity and the aggregation status of the photosensitizer.     

Endogenous protoporphyrin IX, a clinically useful photosensitizer for photodynamic therapy.

The tissue photosensitizer protoporphyrin IX (PpIX) is an immediate precursor of heme in the biosynthetic pathway for heme. In certain types of cells and tissues, the rate of synthesis of PpIX is determined by the rate of synthesis of 5-aminolevulinic acid (ALA), which in turn is regulated via a feedback control mechanism governed by the concentration of free heme. The presence of exogenous ALA bypasses the feedback control, and thus may induce the intracellular accumulation of photosensitizing concentrations of PpIX. However, this occurs only in certain types of cells and tissues. The resulting tissue-specific photosensitization provides a basis for using ALA-induced PpIX for photodynamic therapy. The topical application of ALA to certain malignant and non-malignant lesions of the skin can induce a clinically useful degree of lesion-specific photosensitization. Superficial basal cell carcinomas showed a complete response rate of approximately 79% following a single exposure to light. Recent preclinical studies in experimental animals and human volunteers indicate that ALA can induce a localized tissue-specific photosensitization if administered by intradermal injection. A generalized but still quite tissue-specific photosensitization may be induced if ALA is administered by either subcutaneous or intraperitoneal injection or by mouth. This opens the possibility of using ALA-induced PpIX to treat tumors that are too thick or that lie too deep to be accessible to either topical or locally injected ALA.     

Synthesis of fluorine analogues of protoporphyrin potentially useful for diagnosis and therapy of tumors

With the aim of obtaining a porphyrin derivative useful for diagnosis and therapy of cancer, fluorine analogues of protoporphyrin, in which the vinyl group(s) were replaced by difluorovinyl group(s), were synthesized by the reaction of the formylporphyrins with sodium chlorodifluoroacetate in the presence of triphenylphosphine. Some improvements in the reported procedures for the synthesis of formylporphyrins are also described. Preliminary results of biological tests of the products showed that 8(2),8(2)-difluoroprotoporphyrin accumulates to gastric cancer more selectively than other fluorine analogues and that 3(2),3(2),8(2),8(2)-tetrafluoroprotoporphyrin is taken up by rat hepatoma cells more readily than the others.     

Synthesis of Sn nanocluster@carbon dots for photodynamic therapy application

Recently, photodynamic therapy (PDT) has been extensively applied in clinical and coadjuvant treatment of various kinds of tumors. However, the photosensitizer (PS) of PDT still lack of high production of singlet oxygen (¹O₂), low cytotoxicity and high biocompatibility. Herein, we propose a facile method for establishing a new core-shell structured Sn nanocluster@carbon dots (CDs) PS. Firstly, Sn⁴⁺@S-CDs complex is synthesized using the sulfur-doped CDs (S-CDs) and SnCl₄ as raw materials, and subsequently the new PS (Sn nanocluster@CDs) is obtained after vaporization of Sn⁴⁺@S-CDs solution. Remarkably, the obtained Sn nanocluster@CDs show an enhanced fluorescence as well as a higher ¹O₂ quantum yield (QY) than S-CDs. The high ¹O₂ QY (58.3%) irradiated by the LED light (400–700 nm, 40 mW/cm²), induce the reduction of 4T1 cancer cells viability by 25%. More intriguingly, no visible damage happens to healthy cells, with little impact on liver tissue due to renal excretion, both in vitro and in vivo experiments demonstrate that Sn nanocluster@CDs may become a promising PS, owning a high potential for application in PDT.     

Squaraine dyes for photodynamic therapy: study of their cytotoxicity and genotoxicity in bacteria and mammalian cells

Halogenated squaraine dyes are characterized by long wavelength absorption (>600 nm) and high triplet yields and therefore represent new types of photosensitizers that could be useful for photodynamic therapy. We have analyzed the cytotoxicity and genotoxicity of the bromo derivative 1, the iodo derivative 2 and the corresponding nonhalogenated dye 3 in the absence and presence of visible light. At concentrations of 1-2 microM, 1 and 2 reduced the cloning efficiency of AS52 Chinese hamster ovary cells to less than 1% under conditions that were well tolerated in the dark. Similarly, the proliferation of L5178Y mouse lymphoma cells was inhibited by photoexcited 1 and 2 with high selectivity. The squaraine 3 was much less efficient. Both 1 and 2 induced only few mutations in the gpt locus of the AS52 cells in the presence of light and were not mutagenic in the dark. No mutagenicity with and without irradiation was observed in Salmonella typhimurium TA100 and TA2638. However, both 1 and 2 plus light increased the frequency of micronuclei in AS52 cells. The results indicate that halogenated squaraines exhibit photobiological properties in vitro that are favorable for photodynamic therapeutical applications.     

Topical application of temoporfin-loaded invasomes for photodynamic therapy of subcutaneously implanted tumours in mice: a pilot study

Temoporfin (mTHPC) represents a very potent second-generation synthetic photosensitizer. It has shown to be effective in the photodynamic therapy of early or recurrent oral carcinomas, in the palliative treatment of refractory oral carcinomas and in the treatment of primary non-melanomatous tumours of the skin of the head and neck. Until now for all positive findings an intravenous application of the photosensitizer was mandatory. In the case of cutaneous malignant or non-malignant diseases a topical application of the drug onto the site of the disease followed by illumination, would be advantageous. Unfortunately, mTHPC is a highly hydrophobic drug with a low percutaneous absorption. The purpose of this experiment was to investigate the photodynamic efficacy of novel mTHPC-loaded invasomes after their topical application onto the skin of mice bearing the subcutaneously implanted human colorectal tumour HT29 followed by photoirradiation. Invasomes are vesicles containing in addition to phospholipids a mixture of terpenes (cineole, citral and d-limonene) or only one terpene (citral) and ethanol, as penetration enhancers. This was a pilot study since until now no data are available about the efficacy of mTHPC in the photodynamic therapy of HT29 tumours after its topical application. The aim of this experiment was to investigate whether a mTHPC-loaded invasome formulation can reduce tumour size by photodynamic therapy or at least to find a formulation slowing down tumour growth compared to the control group (mice without any treatment). The groups of mice treated with mTHPC–invasomes containing 1% of the terpene mixture prior to photoirradiation showed a significantly smaller (p < 0.05) tumour increase compared to control groups (mice without any treatment and mice only photoirradiated).     

在光动力光疗中类卟啉稀土配合物光敏作用原初反应的特征: 产生自由基I型机制的ESR研究

本文用ESR方法研究了类卟啉稀土配合物[(CO2H-APPC)Gd]Cl2的光敏反应。用4-hydro-tetramethylpiperidine-N-oxide radical(4-hydro-TEMPO)作探针, 通过对其消自旋的作用, 证实[(CO2H-APPC)Gd]Cl2光敏反应中有阳离子自由基[(CO2H-APPC)Gd]^+产生, 加入还原剂可促使[(CO2H-APPC)Gd]^+生成。经由5, 5-Dimethyl-1-pyrrolineN-oxide(DMPO)对超氧阴离子(O2^-)和羟基自由基(.OH)的自旋捕捉及对该自旋加合物[DMPO-O2^-]和[DMPO-OH]的ESR测定, 证实有O2^-和.OH产生, 并用SOD清除O2^-和甲酸钠清除.OH的实验, 进一步证实O2^-和.OH的产生。上述结果说明[(CO2H-APPC)Gd]Cl2光敏反应存在着产生[(CO2H-APPC)Gd]^+和活性氧自由基的I型机制。     

Ratiometric singlet oxygen nano-optodes and their use for monitoring photodynamic therapy nanoplatforms

Ratiometric photonic explorers for bioanalysis with biologically localized embedding (PEBBLE) nanoprobes have been developed for singlet oxygen, using organically modified silicate (ORMOSIL) nanoparticles as the matrix. A crucial aspect of these ratiometric singlet-oxygen fluorescent probes is their minute size. The ORMOSIL nanoparticles are prepared via a sol-gel-based process and the average diameter of the resultant particles is about 160 nm. These sensors incorporate the singlet-oxygen-sensitive 9,10-dimethyl anthracene as an indicator dye and a singlet-oxygen-insensitive dye, octaethylporphine, as a reference dye for ratiometric fluorescence-based analysis. We have found experimentally that these nanoprobes have much better sensitivity than does the conventional singlet-oxygen-free dye probe, anthracene-9,10-dipropionic acid disodium salt. The much longer lifetime of singlet oxygen in the ORMOSIL matrix, compared to aqueous solutions, in addition to the relatively high singlet oxygen solubility because of the highly permeable structure and the hydrophobic nature of the outer shell of the ORMOSIL nanoparticles, results in an excellent overall response to singlet oxygen. These nanoprobes have been used to monitor the singlet oxygen produced by "dynamic nanoplatforms" that were developed for photodynamic therapy. The singlet oxygen nanoprobes could potentially be used to quantify the singlet oxygen produced by macrophages.     

Lipophilic phthalocyanines for their potential interest in photodynamic therapy: synthesis and photo-physical properties

Several phthalocyanines with different peripheral substituents were prepared and characterized by MALDI-TOF, ¹H NMR, UV–vis, fluorescence, and singlet oxygen quantum yields and retention time in HPLC normal phase. Zinc was used as a central metal ion to increase the photodynamic therapy efficiency. Phthalonitrile or 4-nitro phthalonitriles were used as starting materials. The influence of lipophilicity on the photophysical and photochemical properties was evaluated.     

Progress on the syntheses of fluorine analogs of natural porphyrins potentially useful for the diagnosis and therapy of certain cancers

Hematoporphyrin derivative (HpD) or photofrin II is used as a photosensitizer of photodynamic therapy (PDT) of cancer. However, these are complex mixtures of porphyrin derivatives. We have synthesized fluorine analogs of naturally important porphyrin derivatives, such as protoporphyrin (PP) and hematoporphyrin (HP) for application for the diagnosis and therapy of cancer. Some of these fluorine analogs were found to localize to tumor tissues and/or be taken up selectively by tumor cells. In this review, our former results are outlined briefly, then our recent studies on synthesis of chiral fluorine analogs of HP and synthesis of fluorine analogs of PP using our new zinc reagents will be discussed.     

Complexes of ferromagnetic fluids with photoditazin and their promising applications in photodynamic therapy

Nanosized complexes of ferroparticles with a sensitizer photoditazin (used in photodynamic therapy in oncology) have been synthesized and studied by small-angle neutron scattering. The structure of the complexes was determined, and the influence of the biocompatible polymer pluronic investigated. Clinical tests revealed increased efficiency of the complexes compared with the starting photoditazin. The complexes can be used as the first magnetically operated sensitizer for treatment of oncological diseases by photodynamic therapy.     

Development of new synthetic reactions for nitrogen-containing compounds and their application

Nitrogen-containing compounds are core parts not only of natural and synthetic medicines but also of biologically active compounds including natural products. This review focuses on the development of new synthetic reactions for nitrogen-containing compounds via three methodologies: the reductive photocycloaddition reaction of enamides, radical addition reaction, and nucleophilic addition reaction. Newly developed reactions were successfully applied to the synthesis of various types of nitrogen-containing compounds including medicines, lead compounds to new drugs, natural alkaloids, and others.     

Longer term assessment of photodynamic therapy for intimal hyperplasia: a pilot study

PURPOSE: To determine the potential long term (three or six months) effectiveness of photodynamic therapy (PDT) in reducing intimal hyperplasia in swine. METHODS: Intimal hyperplasia in the abdominal aortae of swine was created by a combination of fat-supplemented diet and balloon catheter injury prior to PDT. Swine were randomly allocated into one of three groups which received either: (i) both drug and light (PDT), (ii) drug only, or (iii) light only. Twenty-four hours following administration of the photosensitizer PHOTOFRIN (porfimer sodium) at 2.5 mg/kg, two distinct 1 cm spots on the posterior wall of the abdominal aorta were illuminated by an argon pumped dye laser tuned to 630 nm for an energy fluence of 120 J/cm2. After three or six months, swine were sacrificed, perfusion fixed, and had their aortae removed for light and electron microscopy. RESULTS: Intimal hyperplasia reduction following PDT persisted for the three or six months follow up period. Experimental vessels receiving PDT showed a 26.0+/-4.5% ( n = 2, ie. four spots) and 30.8+/-5.4% ( n = 1, ie. two spots) smaller percent intimal area after three or six months of recovery, respectively. Control groups receiving either light or drug only showed less than a 6% difference in percent intimal area. Medial and adventitial layers were unaffected in all groups. Electron microscopy demonstrated that the endothelium or endothelial-like cells had regenerated in both the posterior and adjacent areas of the abdominal aortae with no clear difference between them. CONCLUSIONS: These findings suggest that PDT may be beneficial in reducing intimal hyperplasia for up to three or six months in swine.     

Two-photon excitation studies of hypocrellins for photodynamic therapy

The photophysical and photochemical properties of hypocrellins (HA and HB) are examined with two-photon excitations at 800 nm using femtosecond pulses from a Ti:sapphire laser. The two-photon excited fluorescence spectra of HA and HB are very similar to those obtained by one-photon excitation, which may indicate that the two-photon induced photodynamic processes of hypocrellins are similar to one-photon induced photodynamic processes. The two-photon excitation cross sections of HA and HB are measured at 800 nm as about 34.8 x 10(-50) cm(4) s/photon and 21.3 x 10(-50) cm(4) s/photon, respectively. The large two-photon cross sections of both HA and HB, suggest that the hypocrellins can be potential two-photon phototherapeutic agents. As an example for two-photon photodynamic therapy of hypocrellins, we also further examine the cell-damaging effects of HA upon two-photon illumination. Our preliminary results of cell viability test indicate hypocrellins can effectively damage the Hela cells under two-photon illumination.     

Azodioxy compounds as precursors for C-radicals and their application in thermal styrene difunctionalization

An atom-economic thermal α,β-difunctionalization of various styrenes with readily prepared azodioxy compounds is reported. Mechanistic studies reveal that the starting azodioxy compounds can thermally be cleaved to the corresponding C-nitroso compounds, which under these thermal conditions further homolyze to generate reactive C-radicals along with the persistent NO radical. In the presence of a styrene, C-radical addition with subsequent nitrosylation followed by tautomerization is occurring, resulting in an overall styrene β-alkylation-α-oximation reaction.

An atom-economic thermal α,β-difunctionalization of various styrenes with readily prepared azodioxy compounds is reported.     

A first comparative study of purpurinimide-based fluorinated vs. nonfluorinated photosensitizers for photodynamic therapy

A first report on the synthesis and comparative in vitro-in vivo photosensitizing efficacy of various fluorinated and the corresponding nonfluorinated, purpurinimide-based photosensitizers is discussed. In preliminary in vivo screening, compared with the nonfluorinated analogs, purpurinimides bearing trifluoromethyl substituents showed enhanced photosensitizing efficacy. Among compounds (isomers) with similar lipophilicity, the position of the substituents was found to play a decisive role in biological efficacy.     

Metal chelates of porphyrin derivatives as sensitizers in photooxidation processes of sulfur compounds and in photodynamic therapy of cancer

A number of porphyrin derivatives based on hematoporphyrin, 5,10,15,20-tetrasubstituted porphyrins, phthalocyanines, and naphthalocyanines were prepared either as low-molecular compounds or bonded with methoxypoly(ethylene glycol) or attached to silica of low surface area. The low-molecular weight and the polymer-bonded porphyrins exhibit comparable triplet lifetimes and activities in the photosensitized formation of singlet oxygen. For photoninduced processes, the monomeric state of sensitizers is fundamentally important. The porphyrins have been investigated as sensitizers for photooxidation of thiolates and sulfides, which occursvia singlet oxygen, and, therefore, is much more efficient than the corresponding catalytic dark oxidation. Polymer-bonded porphyrins and long-wavelength absorbing naphthalocyanines incorporated in liposomes exhibitin vivo high accumulation in tumor tissues. Under irradiation, singlet oxygen is produced, and efficient phototherapeutic effects are observed, which may be used for photodynamic cancer therapy.Translated fromIzvestiya Akademii Nauk. Seriya Khimicheskaya, No. 12, pp. 2071–2082, December, 1994.D. Wöhrle is very thankful to G. Schulz-Ekloff (Bremen), M. Shopova (Sofia), I. Okura (Tokyo), B. Roeder (Berlin), G. Graschew (Heidelberg), and their coworkers for fruitful cooperation. The research was supported by the German Research Society (Deutsche Forschungsgemeinschaft, 436 BUL 113/51/0, Wo 237/ 12-1), the German Cancer Service (Deutsche Krebshilfe, W 15/89/Sch12), Bremen University (FSP 2/90), and the Federal Department of Scientific Research and Tech nology (Bundesministerium far Forschung und Technotogie, Bonn, 13N6290).     

Recent advances of AIE light-up probes for photodynamic therapy

As a new non-invasive treatment method, photodynamic therapy (PDT) has attracted great attention in biomedical applications. The advantages of possessing fluorescence for photosensitizers have made it possible to combine imaging and diagnosis together with PDT. The unique features of aggregation-induced emission (AIE) fluorogens provide new opportunities for facile design of light-up probes with high signal-to-noise ratios and improved theranostic accuracy and efficacy for image-guided PDT. In this review, we summarize the recent advances of AIE light-up probes for PDT. The strategies and principles to design AIE photosensitizers and light-up probes are firstly introduced. The application of AIE light-up probes in photodynamic antitumor and antibacterial applications is further elaborated in detail, from binding/targeting-mediated, reaction-mediated, and external stimuli-mediated light-up aspects. The challenges and future perspectives of AIE light-up probes in the PDT field are also presented with the hope to encourage more promising developments of AIE materials for phototheranostic applications and translational research.

AIE fluorogens provide new opportunities for the development of light-up probes for photodynamic therapy.