Oxovanadium(IV)-based near-IR PDT agents: design to biological evaluation

An oxovanadium(IV) complex of dipyridophenazine, as a potent metal-based PDT agent, shows efficient DNA photocleavage activity at near-IR region and high photocytotoxicity in both UV-A and visible light in HeLa cells.

Planar triazinium cations from VO2+-assisted ring cyclizations: a remarkably efficient thiazole species for nuclear staining, PDT and anaerobic photocleavage of DNA

Planar triazinium cationic species, from VO2+-assisted cyclization of 1-(2-thiazolylazo)-2-naphthol, shows efficient DNA intercalative binding, visible light-induced anaerobic plasmid DNA photocleavage activity and photocytotoxicity in HeLa and MCF-7 cancer cells by an apoptotic pathway with selective localization of the compound in the nucleus as evidenced from the nuclear staining and confocal imaging.

The experimental research of R-phycoerythrin subunits on cancer treatment: A new photosensitizer in PDT

The mouse tumor cell 5180 and human liver carcinoma cell SMC 7721 cells were first treated with R-PE and its subunits (alpha, beta, gamma subunits), then irradiated with Argon laser (496 nm, 28.8 J/cm(2)). Survival rate was measured by MTT method. In order to compare the phototoxicity in normal cells, the mouse marrow cells were treated with photofrin II and beta-subunit, irradiated with 45 J/cm(2) of light; survival rate was also measured by MTT method. The result showed that R-PE subunits had better PDT effect on s180 cells than R-PE and lower phototoxicity in marrow cells than photofrin II Flow cytometric analysis showed that PDT results in a growth inhibition and a G(0)-G(1) cell cycle arrest in SMC 7721 cells. The tumor cells inhibited by PDT in vivo were morphologically observed by TEM, the tumor cell death was daze to the occlusion of tumor blood vessels and inducement of cell programmed death in nuclei. Therefore, with the advantage in special fluorescence activity, loth molecular weight, good light absorbent character and weak phototoxicity, R-PE subunit is art attractive option for improving the selectivity of PDT.

高效液相色谱法分离测定Fe(Ⅱ)和络合物Fe(Ⅱ)-PDT的两种几何异构体

铁在自然界中广泛分布，其环境和生态效应主要取决于它们的理化性质、形态、溶解性以及与溶解有机质的结合程度。氧化还原性敏感元素铁在自然界主要以Fe(Ⅱ) 和Fe(Ⅲ)两种价态存在。铁在水环境中的地球化学循环作用主要通过其自身的各种价态之间转化以及与周围其他物质(微量元素,营养盐,有机质)之间的相互作用来实现的。不同形态的铁具有不同的理化性质，其环境效应和生物有效性都是不同，不同形态的铁还可对其他重金属在水体中的循环迁移、赋存形态和生物有效性等均产生不同程度的一定的影响。因此，为铁的环境地球化学行为提供更为准确的信息，就必须进行铁的形态分析，由于Fe(Ⅱ)的氧化还原敏感性，故对铁进行形态分析的前提就是准确测定Fe(Ⅱ)的含量，用普通分光光度法测定Fe(Ⅱ)特别是低含量的Fe(Ⅱ)时，结果的不准确性以及高检出限等缺点都严重制约了准确测定环境中Fe(Ⅱ)的含量。为克服用普通分光光度法测定Fe(Ⅱ)时易受样品溶液其他组分的基体干扰以及灵敏度低等缺点，我们采用了用3-(2-吡啶基)-5,6-二苯基-1,2,4-三嗪（PDT）作为柱前衍生试剂，反相离子对高效液相色谱法来测定Fe(Ⅱ)。由于络合物Fe(II)-PDT存在两种几何异构体，故在建立反相离子对高效液相色谱法来测定Fe(Ⅱ)的同时，我们进一步研究了PDT及Fe(II)-PDT的两种几何异构体色谱分离与测定的实验研究，取得了如下进展： 1．研究了有机萃取分光光度法测定Fe(Ⅱ)，实验中发现加入离子对试剂四溴酚酞乙酯钾(TPBE-K)，由于TPBE和Fe(Ⅱ)-PDT络合物生成三元离子缔合物，导致最大特征吸收波长红移，由555nm增加至610nm，三元离子缔合物[Fe(PDT)3] [TBPE]2的摩尔吸收系数由2.4×104 l•mol-1•cm-1增加至1.9×105 l•mol-1•cm-1，显著改善了方法的灵敏度，降低了检出限，在Fe(Ⅱ)含量为0~0.3μg/ml范围内，方法的线性关系良好，线性方程为y＝0.3582x＋0.01，R2＝0.9974，检出限为0.96ng/ml(S/N＝3)，达到ng/ml水平，成功应用于测定泉水及表层湖水的Fe(Ⅱ)及总铁含量。 2．将3-(2-吡啶基)-5,6-二苯基-1,2,4-三嗪(PDT)作为衍生显色络合试剂应用于分光光度法来测定Fe(Ⅱ)已得到较为充分的报道。在上述研究基础上，首次研究了将PDT作为柱前衍生络合试剂，应用于反相离子对高效液相色谱法来测定Fe(Ⅱ)，不仅克服了常规络合衍生分光光度法过量络合剂干扰试验结果的缺点，而且进一步优化选择了检测波长，由于HPLC的应用，突破了在分光光度法中只能在可见波长范围内选择检测波长为555nm，将检测波长选择在Fe(Ⅱ)-PDT吸收更强烈的紫外区295nm处，使得灵敏度得到了显著提高。在Fe(Ⅱ)浓度为0～3.5×10-6mol/l时，峰面积A和峰高H均和标准溶液的浓度C(×10-7mol/l)的线性关系良好,线性回归方程分别为：A=7.4951x-0.0785(R2=0.9999) 和H=0.5126x- 0.0915(R2=0.9997)，方法的检测限为0.35ng/ml (S/N＝3)。本方法测定Fe(Ⅱ)时，不需复杂的前处理步骤，如固相萃取、有机溶液萃取等，直接柱前衍生HPLC方法测定，方便、快速且灵敏度高，低于众多分光光度报道的检出限水准7～170ng/ml范围。 3．由于络合物Fe(II)-PDT存在面式和经式两种几何异构体，故在建立起反相离子对高效液相色谱法来测定Fe(Ⅱ)新方法的同时，进一步研究了PDT及Fe(II)-PDT的两种几何异构体色谱分离及测定实验研究，关于这方面的试验研究，目前来说还没有相关的研究报道，所以将反相离子对高效液相色谱法应用到分离及测定PDT及Fe(II)-PDT的两种几何异构体中，这也是本论文的第二个创新之处。在本实验中，所用的色谱固定相为普通的反相键合C8柱，但由于优化选择了最佳流动相条件，使得PDT及Fe(II)-PDT的两种几何异构体直接在C8柱上得到完全的基线分离。讨论了流动相中有机改性剂乙腈不同比例、不同种类的离子对试剂(高氯酸钠和十二烷基硫酸钠)及不同浓度对PDT和Fe(II)-PDT两种几何异构体的色谱行为的影响。根据不同试验条件下，对所获得的色谱参数(容量因子、分离度、选择性因子、保留时间等)进行了较为深入的分析，据此探讨了色谱分离异构体过程的机理。 4．在上述实验的基础之上，引入更能灵活的调节洗脱强度和被分离物质分离度的三元流动相(乙腈/甲醇/水=20/50/30)，优化选择了三元流动相中有机改性剂的比例以及离子对试剂的种类及浓度，使得异构体的分离度和选择性因子均得到满意结果。对一系列不同浓度的Fe(II)-PDT异构体分离，面式和经式异构体的面积和浓度之间的线性关系在4~25×10-7mol/l范围内良好，线性关系分别为：y = 3.6333x - 2.3378(R2 = 0.9957，经式异构体)，y = 1.8219x - 1.0783(R2 = 0.9964, 面式异构体)。面式和经式异构体的检测限分别为4.28ng/ml和3.44ng/ml (S/N＝3)。 5．进一步研究发现Fe(II)-PDT的两种几何异构体之间存在相互转化的动态平衡，故根据异构体含量随时间的变化就可以探讨在转变过程中的动力学和热力学参数。用高效液相色谱法研究了异构体转变过程中色谱峰高和面积随时间的变化，并依此首次得出了两种几何异构体相互转化可逆过程中的热力学和动力学参数，如熵变、焓变、自由能变化、反应速率常数等，对两种几何异构体之间的动力学转变平衡机理进行合理的解释提供了有益的参考。各不同温度下(30℃，35℃，40℃，45℃)，反应时间在3倍半衰期以内，两种几何异构体之间的相互转变平衡均符合动力学一级反应。各不同温度下Xeln[(Xe-X0)/Xe-X)]随时间变化的线性关系良好，分别为：y=0.0821x+0.7288(R2=0.9911,T=45℃)；y = 0.0486x + 0.598(R2 = 0.9987, T=40℃); y = 0.0216x + 0.5861(R2 = 0.9987, T=35℃)；y = 0.0124x + 0.591(R2 = 0.9988, T=30℃)。 首次将PDT作为柱前衍生络合试剂，应用于反相离子对高效液相色谱法来测定环境中的Fe(Ⅱ)，且无需复杂的样品前处理过程(固相萃取，有机溶液萃取等浓缩富积过程)，克服了普通分光光度法检测限高、易受样品基体效应的干扰等缺点；在建立起的高效液相色谱法测定Fe(Ⅱ)的基础上，进一步对络合物Fe(II)-PDT的两种几何异构体色谱分离及动力学转变实验研究，则是对建立起的反相离子对高效液相色谱法测定Fe(Ⅱ)试验研究基础上的进一步延伸和扩展，是对测定Fe(Ⅱ)的高效液相色谱法的机理的进一步认识和探讨。PDT作为柱前衍生络合试剂首次应用于高效液相色谱测定Fe(Ⅱ)为铁的环境地球化学工作者提供了一种灵敏度高的测定Fe(Ⅱ) 的新途径，为环境样品中Fe(Ⅱ)含量的准确测定提供保证，对铁的环境地球化学研究，如铁形态分析、铁的水-沉积物界面行为等提供更翔实可靠的信息。

Pachymedusa dacnicolor tryptophyllin-1 (PdT-1): structural characterization, pharmacological activity and cloning of precursor cDNA.

Tryptophyllins are a heterogenous group of amphibian skin peptides originally identified in skin extracts of Neotropical leaf frogs, Phyllomedusa sp., by chemical means. Until now, biosynthetic precursor structure and biological activity remain unreported. Here we describe the isolation of a novel, post-translationally modified tryptophyllin, Lys-Pro-Hyp-Ala-Trp-Val-Pro.amide (PdT-1), from the skin secretion of the Mexican leaf frog, Pachymedusa dacnicolor. Using a 3'- and 5'-RACE strategy and an in vitro skin cDNA library, the PdT-1-encoding precursor was cloned and found to consist of an open-reading frame of 62 amino acids with a single copy of PdT-1 located towards the C-terminus. A synthetic replicate of PdT-1 was found to be a potent myoactive agent, relaxing mammalian arterial smooth muscle and contracting small intestinal smooth muscle at nanomolar concentrations. PdT-1 is thus the first amphibian skin tryptophyllin to be pharmacologically characterized and the first whose precursor cDNA has been cloned.

PdT-2: a novel myotropic Type-2 tryptophyllin from the skin secretion of the Mexican giant leaf frog, Pachymedusa dacnicolor.

Amphibian skin secretions represent a unique resource for the discovery of new bioactive peptides. Here we report the isolation, structural and functional characterization of a novel heptapeptide amide, DMSPPWHamide, from the defensive skin secretion of the Mexican giant leaf frog, Pachymedusa dacnicolor. This peptide is of unique primary structure and has been classified as a member of the rather heterogenous tryptophyllin-2 (T-2) family of amphibian skin peptides and named P. dacnicolor Tryptophyllin-2 (PdT-2) in accordance. PdT-2 is the first Type 2-tryptophyllin to possess discrete bioactivity. Both natural and synthetic replicates of the peptide were found to contract the smooth muscle of rat urinary bladder, the latter displaying an EC50 of 4 nM.

Novos derivados porfirínicos: síntese e potenciais aplicações em PDT

O trabalho apresentado nesta dissertação descreve estudos desenvolvidos segundo três metodologias para a funcionalização de meso-tetra-arilporfirinas; uma delas baseada na funcionalização com diazo compostos, outra na reacção de Suzuki-Miyaura e a terceira na reacção de metátese. O presente documento é composto por quatro partes distintas. Na primeira parte são feitas considerações gerais sobre propriedades e aplicações de macrociclos porfirínicos. Na segunda parte é apresentado o trabalho realizado na funcionalização de porfirinas com diazo compostos através de reacções de inserção com um catalisador de ródio(II). Este estudo envolveu a preparação de duas porfirinas com grupos O-H e N-H nas posições para dum substituinte meso-fenilo. Os estudos iniciaram-se com a reacção de 5-(4-hidroxifenil)-10,15,20-trifenilporfirinatozinco(II) com diazoacetato de etilo na presença de Rh2(OAc)4. Obteve-se o produto esperado de inserção com bons rendimentos. A reacção de 5-(4-aminofenil)- 10,15,20-trifenilporfirinatozinco(II) com esse diazo composto na presença de Rh2(OAc)4 mostrou um perfil diferente do da reacção com o outro porfirinato. Foram obtidos três produtos, a saber: produto de bis-inserção e dois outros contendo o grupo funcional amida. Este capítulo descreve ainda a síntese de glicoporfirinas e de glicoclorinas através de reacções de inserção e de ciclopropanação envolvendo, respectivamente, os complexos 5-(4-hidroxifenil)- 10,15,20-trifenilporfirinatozinco(II) e 5,10,15,20- tetraquis(pentafluorofenil)porfirinatozinco(II) com α-diazoacetatos com substituintes sacarídicos. Foram usados, como catalisadores, Rh2(OAc)4 e CuCl, respectivamente, na reacção de inserção e na de ciclopropanação. Tendo em vista a sua aplicação em PDT, os novos produtos foram descomplexados e a unidade glicosídica foi desprotegida. Estudos de geração de oxigénio singuleto mostraram que os compostos obtidos são bons geradores dessa espécie citotóxica. Foram também realizados estudos de avaliação da actividade fotodinâmica em células humanas tumorais HeLa e em células humanas saudáveis HaCaT. Estudos de viabilidade celular, após tratamento fotodinâmico, mostraram que as glicoclorinas são mais eficazes na fotoinactivação das células tumorais. Apenas o derivado da clorina com a unidade de galactose mostrou selectividade para a linha celular tumoral, inibindo o crescimento desta e não causando efeito significativo na linha celular saudável. Este fotossensibilizador não foi tóxico na ausência de luz e depois do tratamento fotodinâmico e em condições sub-letais, provocou vacuolização citoplasmática e retracção pronunciada nas células tumorais, enquanto que nas células saudáveis os danos sofridos foram escassos. Nos estudos de localização celular este fotossensibilizador localizou-se na membrana citoplasmática e nos lisossomas tanto nas células HeLa como nas HaCaT. Na terceira parte deste trabalho são descritos os resultados obtidos nos estudos de funcionalização de porfirinas através da reacção de Suzuki-Miyaura. O complexo 2-(4,4,5,5,-tetrametil-1,3,2-dioxaborolan-2-il)-5,10,15,20- tetrafenilporfirinatozinco(II) foi o composto escolhido como reagente de partida. Usando a reacção de Suzuki foi possível sintetizar complexos β-bromoarilporfirínicos em bons rendimentos. Estes complexos foram sujeitos à reacção de Suzuki com pinacolborano, tendo sido possível obter os produtos de acoplamento esperados e ainda derivados diméricos de complexos porfirínicos ligados por unidades fenileno. Esta metodologia reacional, catalisada por paládio, permitiu ainda sintetizar novos derivados quinolona-porfirina através da reacção do complexo porfirínico β-borilado anteriormente referido com bromo-quinolonas Nsubstiuídas com grupos alquílicos e glicosídicos. Deste modo foram obtidos derivados quinolona-porfirina em bons rendimentos. Com vista a realizar estudos de potencial aplicação em PACT, foram hidrolisados os grupos éster da unidade de quinolona e clivados os grupos protectores das unidades glicosídicas desses derivados. No estudo por MS Tandem (MS/MS) foi possível verificar que os isómeros dos derivados quinolona-porfirina sofrem as mesmas vias de fragmentação, confirmando as suas estruturas análogas. Foi possível ainda distinguir os derivados em que a unidade de porfirina está ligada à quinolona através da posição C-6 da quinolona daqueles em que a unidade de porfirina está ligada à quinolona através da sua posição C-7. Os estudos de geração de oxigénio singuleto mostraram que as porfirinas β- substituídas com unidades de quinolona são melhores geradoras desta espécie citotóxica do que a tetrafenilporfirina e que a eficiência que têm em gerar essa espécie é afectada pela posição da ligação entre a porfirina e a quinolona, assim como com o tipo do N-substituinte da quinolona. As bases livres dos conjugados quinolona-porfirinatos foram testadas como fotossensibilizadores em PACT em células do parasita Leishmania Braziliensis, tendo-se observado que estes compostos têm capacidade para fotoinactivar este parasita. Na quarta parte deste trabalho são descritos os resultados dos estudos de funcionalização de porfirinas através da reacção de metátese com catalisador de Grubbs, visando a obtenção de novos macrociclos com grupos triazolilo. Verificouse que a eficiência da reacção de metátese cruzada entre 2-vinil-5,10,15,20- tetrafenilporfirinatozinco(II) e 4-vinil-1,2,3-triazóis N-substituídos dependia das condições reaccionais e do triazol usado em cada caso. Foi possível ainda concluir que o impedimento estéreo das espécies envolvidas é um dos responsáveis pelo ciclo catalítico prosseguir por uma via secundária, originando uma espécie de ruténio pouco eficiente como catalisador. Este estudo foi estendido à reacção dos 4-vinil-1,2,3-triazois seleccionados com 2-butadienil-5,10,15,20- tetrafenilporfirinatoniquel(II); em cada caso foi possível obter uma mistura de isómeros dos derivados triazol-porfirina, geralmente em maiores rendimentos globais do que na reacção com 2-vinil-5,10,15,20-tetrafenilporfirinatozinco(II). Este facto será indicativo de que a reacção de metátase com esse derivado porfirínico segue, predominantemente, a via mais eficiente.

Phthalocyanines : interaction with carbon structures and as PDT agents

This dissertation describes the synthesis and characterization of different phthalocyanine (Pc) derivatives, as well as some porphyrins (Pors), for supramolecular interaction with different carbon nanostructures, to evaluate their potential application in electronic nanodevices. Likewise, it is also reported the preparation and biological evaluation of interesting phthalocyanine conjugates for cancer photodynamic therapy (PDT) and microorganisms photodynamic inactivation (PDI). The phthalonitrile precursors were prepared from commercial phthalonitriles by nucleophilic substitution of -NO2, -Cl, or -F groups, present in the phthalonitrile core, by thiol or pyridyl units. After the synthesis of these phthalonitriles, the corresponding Pcs were prepared by ciclotetramerization using a metallic salt as template at high temperatures. A second strategy involved the postfunctionalization of hexadecafluorophthalocyaninato zinc(II) through the adequate substituents of mercaptopyridine or cyclodextrin units on the macrocycle periphery. The different compounds were structurally characterized by diverse spectroscopic techniques, namely 1H, 13C and 19F nuclear magnetic resonance spectroscopies (attending the elemental composition of each structure); absorption and emission spectroscopy, and mass spectrometry. For the specific photophysical studies were also used electrochemical characterization, femtosecond and raman spectroscopy, transmission electron and atomic force microscopy. It was highlighted the noncovalent derivatisation of carbon nanostructures, mainly single wall carbon nanotubes (SWNT) and graphene nanosheets with the prepared Pc conjugates to study the photophysical properties of these supramolecular nanoassemblies. Also, from pyridyl-Pors and ruthenium phthalocyanines (RuPcs) were performed Por-RuPcs arrays via coordination chemistry. The results obtained of the novel supramolecular assemblies showed interesting electron donor-acceptor interactions and might be considered attractive candidates for nanotechnological devices. On the other hand, the amphiphilic phthalocyanine-cyclodextrin (Pc-CD) conjugates were tested in biological trials to assess their ability to inhibit UMUC- 3 human bladder cancer cells. The results obtained demonstrated that these photoactive conjugates are highly phototoxic against human bladder cancer cells and could be applied as promising PDT drugs.

Analysis of the combined effect of lasers of different wavelengths for PDT outcome using 600, 630, and 660 nm

We investigated the effects of photodynamic therapy (PDT) outcome when combining three laser systems that produce light in three different wavelengths (600, 630, and 660 nm). Cooperative as well as independent effects can be observed. We compared the results of the combined wavelengths of light with the effect of single laser for the excitation of the photosensitizer. In the current experiment, the used photosensitizer was Photogem (R) (1.5 mg/kg). Combining two wavelengths for PDT, their cumulative dose and different penetrability may change the overall effect of the fluence of light, which can be effective for increasing the depth of necrosis. This evaluation was performed by comparing the depth and specific aspect of necrosis obtained by using single and dual wavelengths for irradiation of healthy liver of male Wistar rats. We used 15 animals and divided them in five groups of three animals. First, Photogem (R) was administered; follow by measurement of the fluorescence spectrum of the liver before PDT to confirm the level of accumulation of photosensitizer in the tissue. After that, an area of 1 cm(2) of the liver was illuminated using different laser combinations. Qualitative analysis of the necrosis was carried out through histological and morphological study. [GRAPHICS] (a) - microscopic images of rat liver cells, (b) - superficial necrosis caused by PDT using dual-wavelength illumination, (c) - neutrophilic infiltration around the vessel inside the necrosis, and (d) - neutrophilic infiltration around the vessel between necrosis and live tissue (C) 2011 by Astro Ltd. Published exclusively by WILEY-VCH Verlag GmbH & Co. KGaA

A new micro/nanoencapsulated porphyrin formulation for PDT treatment

The highly hydrophobic 5,10,15-triphenyl-20-(3-N-methylpyridinium-yl)porphyrin(3MMe)cationic species was synthesized, characterized and encapsulated in marine atelocollagen/xanthane gum microcapsules by the coacervation method. Further reduction in the capsule size, from several microns down to about 300-400 nm, was carried out successfully by ultrasonic processing in the presence of up to 1.6% Tween 20 surfactant, without affecting the distribution of 3MMe in the oily core. The resulting creamlike product exhibited enhanced photodynamic activity but negligible cytotoxicity towards HeLa cells. The polymeric micro/nanocapsule formulation was found to be about 4 times more phototoxic than the respective phosphatidylcholine lipidic emulsion, demonstrating high potentiality for photodynamic therapy applications. (C) 2009 Elsevier B.V. All rights reserved.

Chemiluminescence as a PDT light source for microbial control

The photodynamic therapy (PDT) is a combination of using a photosensitizer agent, light and oxygen that can cause oxidative cellular damage. This technique is applied in several cases, including for microbial control. The most extensively studied light sources for this purpose are lasers and LED-based systems. Few studies treat alternative light sources based PDT. Sources which present flexibility, portability and economic advantages are of great interest. In this study, we evaluated the in vitro feasibility for the use of chemiluminescence as a PDT light source to induce Staphylococcus aureus reduction. The Photogem (R) concentration varied from 0 to 75 mu g/ml and the illumination time varied from 60 min to 240 min. The long exposure time was necessary due to the low irradiance achieved with chemiluminescence reaction at mu W/cm(2) level. The results demonstrated an effective microbial reduction of around 98% for the highest photosensitizer concentration and light dose. These data suggest the potential use of chemiluminescence as a light source for PDT microbial control, with advantages in terms of flexibility, when compared with conventional sources. (C) 2011 Elsevier B.V. All rights reserved.

The importance of protoporphyrin IX efflux for ALA-PDT dosimetry

One of the major advances in PDT is the use of 5-aminolevulinic acid (ALA) to induce the production of an enclogenous photosensitizer inside the cells using intracellular enzymatic pathways. ALA is the first intermediate in heme biosynthesis and a precursor of the protoporphyrin IX (PpIX). When activated by light, this efficient photosensitizer accumulated in the target cells can produce cytotoxicity. The aim of this study was to find the best conditions for cell killing using ALA to temporarily increase the concentration of PpIX in two cell lines. It was shown that a considerable efflux of synthesized PpIX occurs. Since this efflux is time-dependent, it is essential to know the optimum time for irradiation after ALA administration. So, the efflux of PpIX from the cells is an important parameter to be considered for ALA-PDT dosimetry.