金属酞菁光敏剂的三阶非线性光学性能研究

从光动力治疗癌症的疗效着眼研究酞菁配合物的三阶非线性光学性能。用时间分辨简并四波混频方法测量苯硫基钛菁锌(C56H32S4Zn),苯硫基铝酞菁(C56H32AlN8O4)以及烷氧基铝酞菁(C56H32AlN8O4)的三阶非线性光极化率;测量四波混频响应的衰减过程;研究时间响应的超快过程和慢过程及其动力学机制,它们分别对应于单态和三线态的寿命。在荧光显微成像系统中观察三种酞菁光敏剂对人肝癌细胞杀伤的形态变化,并用MTT方法检测细胞存活率。对三种酞菁配合物的三线态量子产率和寿命进行测定,结果与它们对人肝癌细胞的光动力杀伤作用相关联。     

能量化时线粒体内膜表面电荷的变化

本文报告用荧光探剂1,8—ANS和电泳激光光散射技术,研究鼠肝线粒体内膜在加入ATP的能量化过程中其膜表面电荷的变化。实验结果表明在加入ATP后线粒体内膜的能量化使其膜表面的负电荷减少。作者论讨了用上述二种方法研究线粒体内膜在能量化时表面电荷变化的有关问题。     

酞菁类光敏剂对肝癌细胞线粒体和微粒体的光辐射效应

酞菁(Phthalocyanin,PC)化合物结构类似卟啉,是一种新的光敏剂。它的四个苯环上各取代一个磺酸基成为四磺酸酞菁(phthalocyanine tetrasulfonate, TSPC)。TSPC-30μg/ml合并照光30分钟,肝癌细胞线粒体ATP酶和微粒体G-6-P酶明显受抑,对线粒体单胺氧化酶(MAO)没有明显影响。在上述剂量和照光条件下,线粒体和微粒体膜蛋白巯基含量显著减少,而膜脂质过氧化产物增多,线粒体膜通透性改变,导致线粒体肿胀。     

刀豆球蛋白A对Ehrlich腹水癌细胞的膜电位和表面电荷的影响

本文报告用荧光探剂diS-C_3—(5)和细胞电泳技术研究刀豆球蛋白A(ConA)作用于Ehrlich腹水癌细胞引起的膜电位和表面电荷的变化.ConA与细胞膜相应受体结合,导致在膜上的diS-C_3-(5)的荧光强度增加,表明细胞去极化.经用缬氨霉素诱导的钾扩散电位校正,与光学讯号变化相应的膜电位变化约是20mv.细胞经G毒毛旋化苷处理后,ConA引起的去极化程度比未处理过的细胞大.ConA作用于Ehrlich腹水癌细胞使细胞电泳迁移率变小.表明细胞表面负电荷数目减少.本文对这些变化的可能机制和相互关系进行了讨论.     

脱落酸对植物线粒体膜生物物理特性的影响

采用１,６－二苯基－１,３,５－已三乙烯（ＤＰＨ）荧光偏振法和中性红法分别研究了脱落酸（ＡＢＡ）对玉米黄化芽及大豆子叶离体线粒体膜的微粘度（η）、表面电位（ψ）等生物物理特性的影响。结果表明：ＡＢＡ有降低线粒体膜的微粘度及提高线粒体膜的表面电位作用,并导致呼吸速率升高,呼吸控制和氧化磷酸化偶联下降。ＡＢＡ对线粒体膜微粘度的作用具有浓度饱和效应;ＡＢＡ对线粒体膜表面电位的提高作用,因植物不同而有差异,对玉米的作用要大于对大豆的。追踪线粒体Ａ（３５０）值的变化,还证实,ＡＢＡ提高了大豆线粒体的Ａ（３５０）值,即导致大豆线粒体的相互聚集（ａｇｇｒｅｇａｔｉｏｎ）。     

Mg~(2 )离子对紫膜表面电位效应的自旋探针—ESR研究

本文根据带正电荷自旋探针CAT_(12)在紫膜结合相和水相的分布,利用自旋探针顺磁共振(ESR)技术测定了Mg~(2 )对紫膜表面电位的影响,我们的结果表明紫膜具有σ为3.02×10~(-4)Ch-arges/(?)~2的表面电荷密度.据此σ用Gouy-Chapman理论计算得到Mg~(2 )离子浓度与表面电位((?)_i)的关系与实验结果极为一致,这表明离子通过表面电位的变化引起紫膜的表面pH值的改变从而影响紫膜的结构与功能,Mg~(2 )对紫膜表面电位的影响明显地比K~ 要大,说明镁离子可能在紫膜的结构与功能中有更为重要的作用.     

Mg~(2+)离子对紫膜表面电位效应的自旋探针—ESR研究

本文根据带正电荷自旋探针CAT_(12)在紫膜结合相和水相的分布,利用自旋探针顺磁共振(ESR)技术测定了Mg~(2+)对紫膜表面电位的影响,我们的结果表明紫膜具有σ为3.02×10~(-4)Ch-arges/(?)~2的表面电荷密度.据此σ用Gouy-Chapman理论计算得到Mg~(2+)离子浓度与表面电位((?)_i)的关系与实验结果极为一致,这表明离子通过表面电位的变化引起紫膜的表面pH值的改变从而影响紫膜的结构与功能,Mg~(2+)对紫膜表面电位的影响明显地比K~+要大,说明镁离子可能在紫膜的结构与功能中有更为重要的作用.     

凝集素芯片对体液细胞进行检测方法的建立和初步应用

建立对体液细胞进行自动捕获的凝集素芯片体系,利用凝集素对糖链的特异亲和作用捕获细胞,提取白血病患者外周血、肺癌胸水和肝腹水中细胞进行荧光标记,凝集素芯片捕获,激光扫描仪检测捕获细胞的荧光信号,常规HE染色后光学显微镜下观察细胞的形态并进行免疫化学反应,流式细胞仪验证凝集素芯片的特异性．结果表明：凝集素芯片可以对体液中的癌细胞进行自动捕获,对癌细胞膜表面糖链进行识别．芯片检测的细胞浓度最少可达每mL10^4个左右．芯片有较好的重复性和特异性．这种凝集素芯片可用于临床体液中癌细胞的检测分析,对癌细胞膜表面凝集素亲和位点进行即时、高通量的检测,为了解细胞膜表面聚糖在癌变过程中的变化提供了一个技术平台．     

声波刺激对菊花愈伤组织膜脂物理状态和膜脂代谢的影响

以蔗糖梯度法纯化的菊花 (Dendranthemamorifolium (Ramat.)Tzvel.)愈伤组织质膜微囊为材料 ,研究了声波刺激下质膜膜脂代谢和物理状态的变化。结果表明 ,一定强度 (10 0dB)和频率 (10 0 0Hz)的声波刺激使质膜磷酸二脂含量和二苯己三烯 (DPH)荧光偏振值降低 ,质膜光散射值、MC5 40荧光强度和磷酸单脂含量增加。表明一定强度和频率的声波刺激使质膜变疏松 ,膜的流动性增加 ,膜表面电荷密度和疏水性降低 ,膜脂合成代谢增加 ,分解代谢减弱。由此可见 ,膜脂物理状态和膜脂代谢对声波刺激极为敏感。     

声波刺激对菊花愈伤组织膜脂物理状态和膜脂代谢的影响

以蔗糖梯度法纯化的菊花(Dendranthema morifolium (Ramat.) Tzvel.)愈伤组织质膜微囊为材料,研究了声波刺激下质膜膜脂代谢和物理状态的变化.结果表明, 一定强度(100 dB)和频率(1 000 Hz)的声波刺激使质膜磷酸二脂含量和二苯己三烯(DPH)荧光偏振值降低,质膜光散射值、MC540荧光强度和磷酸单脂含量增加.表明一定强度和频率的声波刺激使质膜变疏松,膜的流动性增加,膜表面电荷密度和疏水性降低,膜脂合成代谢增加,分解代谢减弱.由此可见,膜脂物理状态和膜脂代谢对声波刺激极为敏感.     

Cytotoxicity of PP(Arg)(2)- and Hp(Arg)(2)-mediated photodynamic therapy and early stage of apoptosis induction in prostate carcinoma in vitro

Porphyrin photosensitizers tend to localize in mitochondria. The depolarization of mitochondrial membrane is one of the early stages of apoptosis and Laser Scanning Fluorescence Microscopy allows to determine changes in transmembrane mitochondrial potential under influence of PDT depending on the kind of photosensitizer (PP(Arg)(2), Hp(Arg)(2)), the energy dose (5, 10, 30 and 50 J/cm(2)) and time periods (24 and 48 hours after irradiation) in the LNCaP (lymphonodal metastasis of prostate carcinoma, the androgen dependent cell line). Cyototoxicity induced by PP(Arg)(2)- and Hp(Arg)(2)-based PDT depending on energy dose and time after irradiation in prostate carcinoma is determined with MTT. Generally, it was shown that lower energy doses induce greater changes in transmembrane mitochondrial potential. Hp(Arg)(2)-based PDT was more effective causing greater mitochondrial membrane depolarization and cell viability decrease in comparison to PP(Arg)(2)-mediated PDT (in the case of maximal nontoxic photosensitizer doses used).     

Role of mitochondria in cell death induced by Photofrin-PDT and ursodeoxycholic acid by means of SLIM.

The present study was undertaken to find new ways to improve efficacy of photodynamic therapy (PDT). We investigated the combinatory effect of the photosensitizer Photofrin and ursodeoxycholic acid (UDCA). UDCA is a relatively non-toxic bile acid which is used inter alia as a treatment for cholestatic disorders and was reported to enhance PDT efficiency of two other photosensitizers. Since besides necrosis and autophagic processes apoptosis has been found to be a prominent form of cell death in response to PDT for many cells in culture, several appropriate tests, such as cytochrome c release, caspase activation and DNA fragmentation were performed. Furthermore spectral resolved fluorescence lifetime imaging (SLIM) was used to analyse the cellular composition of Photofrin and the status of the enzymes of the respiratory chain. Our experiments with two human hepatoblastoma cell lines revealed that the combination of Photofrin with UDCA significantly enhanced efficacy of PDT for both cell lines even though the underlying molecular mechanism for the mode of action of Photofrin seems to be different to some extent. In HepG2 cells cell death was clearly the consequence of mitochondrial disturbance as shown by cytochrome c release and DNA fragmentation, whereas in Huh7 cells these features were not observed. Other mechanisms seem to be more important in this case. One reason for the enhanced PDT effect when UDCA is also applied could be that UDCA destabilizes the mitochondrial membrane. This could be concluded from the fluorescence lifetime of the respiratory chain enzymes which turned out to be longer in the presence of UDCA in HepG2 cells, suggesting a perturbation of the mitochondrial membrane. The threshold at which PDT damages the mitochondrial membrane was therefore lower and correlated with the enhanced cytochrome c release observed post PDT. Thus enforced photodamage leads to a higher loss of cell viability.     

Photodynamic therapy regulates fate of cancer stem cells through reactive oxygen species

Photodynamic therapy (PDT) is an effective and promising cancer treatment. PDT directly generates reactive oxygen species (ROS) through photochemical reactions. This oxygen-dependent exogenous ROS has anti-cancer stem cell (CSC) effect. In addition, PDT may also increase ROS production by altering metabolism, endoplasmic reticulum stress, or potential of mitochondrial membrane. It is known that the half-life of ROS in PDT is short, with high reactivity and limited diffusion distance. Therefore, the main targeting position of PDT is often the subcellular localization of photosensitizers, which is helpful for us to explain how PDT affects CSC characteristics, including differentiation, self-renewal, apoptosis, autophagy, and immunogenicity. Broadly speaking, excess ROS will damage the redox system and cause oxidative damage to molecules such as DNA, change mitochondrial permeability, activate unfolded protein response, autophagy, and CSC resting state. Therefore, understanding the molecular mechanism by which ROS affect CSCs is beneficial to improve the efficiency of PDT and prevent tumor recurrence and metastasis. In this article, we review the effects of two types of photochemical reactions on PDT, the metabolic processes, and the biological effects of ROS in different subcellular locations on CSCs.     

Protein damage by photo-activated Zn(II) N-alkylpyridylporphyrins

Destruction of unwanted cells and tissues in photodynamic therapy (PDT) is achieved by a combination of light, oxygen, and light-sensitive molecules. The advantages of PDT compared to other traditional treatment modalities, and the shortcomings of the currently used photosensitizers, have stimulated the search for new, more efficient photosensitizer candidates. Ability to inflict selective damage to particular proteins through photo-irradiation would significantly advance the design of highly specific photosensitizers. Achieving this objective requires comprehensive knowledge concerning the interactions of the particular photosensitizer with specific targets. Here, we summarize the effects of Zn(II) N-alkylpyridylporphyrin-based photosensitizers on intracellular (metabolic, antioxidant and mitochondrial enzymes) and membrane proteins. We emphasize how the structural modifications of the porphyrin side substituents affect their lipophilicity, which in turn influence their subcellular localization. Thus, Zn(II) N-alkylpyridylporphyrins target particular cellular sites and proteins of interest, and are more efficient than hematoporphyrin D, whose commercial preparation (Photofrin) has been clinically approved for PDT.     

Photodynamic Therapy in Pythium insidiosum – An In Vitro Study of the Correlation of Sensitizer Localization and Cell Death

Pythiosis is an infectious disease caused by Pythium insidiosum, a fungus-like organism. Due to the lack of ergosterol on its cell membrane, antibiotic therapy is ineffective. The conventional treatment is surgery, but lesion recurrence is frequent, requiring several resections or limb amputation. Photodynamic therapy uses photo-activation of drugs and has the potential to be an attractive alternative option. The in vitro PDT response on the growing of Pythium insidiosum culture was investigated using three distinct photosensitizers: methylene blue, Photogem, and Photodithazine. The photosensitizer distribution in cell structures and the PDT response for incubation times of 30, 60, and 120 minutes were evaluated. Methylene blue did not penetrate in the pathogen''s cell and consequently there was no PDT inactivation. Photogem showed heterogenous distribution in the hyphal structure with small concentration inside the cells. Porphyrin-PDT response was heterogenous, death and live cells were observed in the treated culture. After 48 hours, hyphae regrowth was observed. Photodithazine showed more homogenous distribution inside the cell and with the specific intracellular localization dependent on incubation time. Photodithazine first accumulates in intracellular vacuoles, and at incubation times of one hour, it is located at all cell membranes. Higher inhibition of the growing rates was achieved with Photodithazine -PDT, over 98%. Our results showed that the photosensitizers that cross more efficiently the Pythium insidiosum membranes are able to cause extensive damage to the organism under illumination and therefore, are the best options for clinical treatment.     

Milestones in the development of photodynamic therapy and fluorescence diagnosis.

Many reviews on PDT have been published. This field is now so large, and embraces so many sub-specialties, from laser technology and optical penetration through diffusing media to a number of medical fields including dermatology, gastroenterology, ophthalmology, blood sterilization and treatment of microbial-viral diseases, that it is impossible to cover all aspects in a single review. Here, we will concentrate on a few basic aspects, all important for the route of development leading PDT to its present state: early work on hematoporphyrin and hematoporphyrin derivative, second and third generation photosensitizers, 5-aminolevulinic acid and its derivatives, oxygen and singlet oxygen, PDT effects on cell organelles, mutagenic potential, the basis for tumour selectivity, cell cooperativity, photochemical internalization, light penetration into tissue and the significance of oxygen depletion, photobleaching of photosensitizers, optimal light sources, effects on the immune system, and, finally, future trends.     

A study on the photodynamic properties of chlorophyll derivatives using human hepatocellular carcinoma cells.

Photodynamic therapy (PDT) is an alternative anticancer treatment in which direct tumor-cell killing results from selective accumulation of photosensitizers in the tumor sites and phototoxicity occurs when light-activated photosensitizers transfer the energy to oxygen nearby to produce singlet oxygen. The objective of this study was to investigate the effects of PDT using chlorophyll derivatives such as pheophytin a (phe a), pheophytin b (phe b), pheophorbide a (pho a) and pheophorbide b (pho b) as the photosensitizers, and the 660 nm light-emitting diodes (LEDs) irradiation on human hepatocellular carcinoma cells (HuH-7). The drug concentration-dependent inhibition of HuH-7 cell viability was studied under LEDs irradiation (10 mW cm(-2)) at radiant exposure of 5.1 and 10.2 J cm(-2) by MTT assay. Significant inhibition of the survival of HuH-7 cells (<10%) was observed when an irradiation dose of 10.2 J cm(-2) combined with the concentration of 0.5 microg ml(-1) of phe a, 0.125 microg ml(-1) of pho a, 0.25 microg ml(-1) of phe b, and 0.125 microg ml(-1) of pho b were applied. The results from Annexin V-propidium iodide staining revealed that phe a, phe b, pho a and pho b could induce cell death in HuH-7 cells predominantly via a necrotic process. The results from immunoblot analyses exhibited that chlorophyll derivative-mediated PDT initiated cytochrome c release, caspase-9 and caspase-3 activation, followed by poly ADP-ribose polymerase (PARP) cleavage. Thus, apoptosis also occurred in HuH-7 cells after PDT treatment, and the execution of the apoptotic process may be initiated from the loss of mitochondrial function. Our findings demonstrate that both apoptosis and necrosis can be induced in HuH-7 cells after PDT using phe a, phe b, pho a and pho b and LEDs.     

Towards PDT with Genetically Encoded Photosensitizer KillerRed: A Comparison of Continuous and Pulsed Laser Regimens in an Animal Tumor Model

The strong phototoxicity of the red fluorescent protein KillerRed allows it to be considered as a potential genetically encoded photosensitizer for the photodynamic therapy (PDT) of cancer. The advantages of KillerRed over chemical photosensitizers are its expression in tumor cells transduced with the appropriate gene and direct killing of cells through precise damage to any desired cell compartment. The ability of KillerRed to affect cell division and to induce cell death has already been demonstrated in cancer cell lines in vitro and HeLa tumor xenografts in vivo. However, the further development of this approach for PDT requires optimization of the method of treatment. In this study we tested the continuous wave (593 nm) and pulsed laser (584 nm, 10 Hz, 18 ns) modes to achieve an antitumor effect. The research was implemented on CT26 subcutaneous mouse tumors expressing KillerRed in fusion with histone H2B. The results showed that the pulsed mode provided a higher rate of photobleaching of KillerRed without any temperature increase on the tumor surface. PDT with the continuous wave laser was ineffective against CT26 tumors in mice, whereas the pulsed laser induced pronounced histopathological changes and inhibition of tumor growth. Therefore, we selected an effective regimen for PDT when using the genetically encoded photosensitizer KillerRed and pulsed laser irradiation.     

Cell death induced by MPPa-PDT in prostate carcinoma in vitro and in vivo

Lack of effective photosensitizers has become a major limit for extensive application of photodynamic therapy. In this study, the photocytotoxicity and mode of death induced by a newly developed photosensitizer MPPa, a derivative of chlorophyll a, were investigated in PC-3M cell line, a highly metastatic variant of poorly differentiated androgen-independent proctanec adenocarcinoma PC-3. MTT reduction assay was used to measure cytotoxicity in both PC-3M and HUVEC, after which a flow cytometer was used to measure apoptotic rate and cell cycle, and then Caspase-3, -8, -9 were investigated. Finally, an animal model was set up to embody the curative effect and for histopathological examinations. The photocytotoxicity of MPPa showed both light- and drug-dose dependent characteristics and no significant dark cytotoxicity was observed in PC-3M cells. In HUVEC, MPPa exhibited an obviously low cytotoxicity. By other in vitro studies, we found MPPa-PDT induced apoptotic mainly via the mitochondrial/Caspase-9/Caspase-3 pathway and could restrain the cell cycle progression from the more sensitive G0/G1-phases. In vivo, the tumour growth was significantly inhibited after PDT, and many apoptotic cells could be seen by histopathological examinations. These results indicate the death way of cells induced by MPPa is mainly via mild apoptotic and the cure effect is obvious, suggesting that MPPa is a potential photosensitizer of photodynamic therapy for prostate cancer.     

New porphyrin amino acid conjugates: Synthesis and photodynamic effect in human epithelial cells

The efficacy of new porphyrin amino acid conjugates as photosensitizers for photodynamic therapy (PDT) were assayed in vitro on tumoral (HeLa) and on non tumoral (HaCaT) human cell lines. The conjugates stable in liposomes are able to penetrate efficiently in the cytoplasm of cultured cancer and normal cells. No dark cytotoxicity is observed at the same concentration used for PDT cell treatment and during long incubation time (24 h). The cell survival after the PDT treatment with visible light is dependent upon light exposure level and compound concentration. The tested compounds show higher photocytotoxicity in tumoral HeLa cells than in no tumoral HaCaT cells. The results suggest that these amino acid porphyrin conjugates are potential photosensitizers for PDT.