Photodynamic performance of amphiphilic chlorin e6 derivatives with appropriate properties: A comparison between different-type liposomes as delivery systems.

BACKGROUND: Many aspects are currently being investigated, with the aim of improving the application of PDT in the clinic by rendering it more effective. One of the current trends focuses on the use of nanocarriers. The aim of this study is to describe novel photosensitizers among polyol amide chlorin e6 derivatives for photodynamic therapy (PDT) using liposomes. METHODS: In addition to their intracellular localization and antiproliferative activity against HCT116 cells, appropriate photophysical features have been determined (especially high 1O2 quantum yield production). RESULTS AND CONCLUSIONS: Fluorescent microscopy demonstrated that the compounds entered the endoplasmic reticulum (ER), lysosomes, mitochondria and partially the cytoplasm. All of the chlorins showed no dark cytotoxicity; however, high phototoxicity was observed. Using optical and electron microscopy, we investigated the impact of chlorin-based PDT upon cell damage leading to cell death. Chl ara 3 was identified as the most promising compound among polyol amide chlorin e6 derivatives and improved phototoxicity was observed as compared with a clinically approved temoporfin. Our results indicate that newly-synthesized chlorins seem to be promising candidates for PDT application, and two of them (chl ara 3 and chl mme 2) may create promising new drugs, both in the form of a free compound and as a liposomal formulation.

Cobalt protoporphyrin IX increases endogenous G-CSF and mobilizes HSC and granulocytes to the blood.

Granulocyte colony-stimulating factor (G-CSF) is used in clinical practice to mobilize cells from the bone marrow to the blood; however, it is not always effective. We show that cobalt protoporphyrin IX (CoPP) increases plasma concentrations of G-CSF, IL-6, and MCP-1 in mice, triggering the mobilization of granulocytes and hematopoietic stem and progenitor cells (HSPC). Compared with recombinant G-CSF, CoPP mobilizes higher number of HSPC and mature granulocytes. In contrast to G-CSF, CoPP does not increase the number of circulating T cells. Transplantation of CoPP-mobilized peripheral blood mononuclear cells (PBMC) results in higher chimerism and faster hematopoietic reconstitution than transplantation of PBMC mobilized by G-CSF. Although CoPP is used to activate Nrf2/HO-1 axis, the observed effects are Nrf2/HO-1 independent. Concluding, CoPP increases expression of mobilization-related cytokines and has superior mobilizing efficiency compared with recombinant G-CSF. This observation could lead to the development of new strategies for the treatment of neutropenia and HSPC transplantation.

Platinum(II) coordination compounds with 4'-pyridyl functionalized 2,2':6',2″-terpyridines as an alternative to enhanced chemotherapy efficacy and reduced side-effects.

Two platinum(II) coordination compounds, [PtCl(4'-R1-terpy)](SO3CF3) (1) and [PtCl(4'-R2-terpy)](SO3CF3) (2), with 4'-(2-pyridyl)-2,2':6',2″-terpyridine (4'-R1-terpy) or 4'-(3-pyridyl)-2,2':6',2″-terpyridine (4'-R2-terpy) were synthesized and the impact of the pendant pyridyl ring on the structure and cytotoxic activity of Pt(II)-terpyridine complexes was explored. The single-crystal X-ray diffraction analysis confirmed square planar coordination of the cations [PtCl(4'-Rn-terpy)]+. The mode of binding of 1 and 2 to calf thymus DNA was examined by UV-Vis absorption titration, ethidium displacement assay and reaction with 9-ethylguanine, and the mixed covalent-intercalative mode was demonstrated. The cytotoxicity of the Pt(II) complexes against six cancer cell lines and three normal ones was determined using MTS (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium) assay and compared to cisplatin. The IC50 values for the compound 2 towards the cancer cell lines are in the low micromolar range. Most remarkably, 2 was over 4 times more effective than 1 and cisplatin against non-small lung adenocarcinoma (A549), and its selectivity index was ~60-80 times higher than that for 1 and cisplatin. The mechanisms underlying the loss of viability under treatment of 2 was further investigated including F-actin staining, mitotic index analysis, cytometric cell cycle analysis, Fluorescein isothiocyanate (FITC) -conjugated Annexin V antibody and propidium iodide (PI) staining, measurements of reactive oxygen species (ROS) in cells, analysis of changes in the mitochondrial mass and potential and quantitative real time polymerase chain reaction (qRT-PCR) genes analysis. The compound 2 was found to have a pro-oxidative effect by strong stimulation of cells for the production of reactive oxygen species and cytostatic effect through cell cycle arrest.

Iron Chelators and Exogenic Photosensitizers. Synergy through Oxidative Stress Gene Expression.

In non-invasive anticancer photodynamic therapy (PDT), a nontoxic photosensitizer (PS), which is activated by visible light, is used as a magic bullet that selectively destroys cancer cells. Recently, we described the combined therapy of 5-aminolevulinic acid (ALA-PDT) with thiosemicarbazone (TSC), i.e. an iron-chelating agent. This resulted in a strong synergistic effect. Herein, we investigated a novel strategy using a combination of PDT consist of the xenobiotic-porphyrin type PS with TSC. We observed a synergistic effect for all of the pairs of TSC-PS. This approach can be rationalized by the fact that both chlorin and TSC can affect the generation of reactive oxygen species (ROS). In order to elucidate the plausible mechanism of action, we also combined the investigated PSs with DFO, which forms complexes that are redox inactive. We detected a slight antagonism or additivity for this combination. This may suggest that the ability of an iron chelator (IC) to participate in the production of ROS and the generation of oxidative stress is important.

New insight into the shortening of the collagen fibril D-period in human cornea.

Collagen fibrils type I display a typical banding pattern, so-called D-periodicity, of about 67 nm, when visualized by atomic force or electron microscopy imaging. Herein we report on a significant shortening of the D-period for human corneal collagen fibrils type I (21 ± 4 nm) upon air-drying, whereas no changes in the D-period were observed for human scleral collagen fibrils type I (64 ± 4 nm) measured under the same experimental conditions as the cornea. It was also found that for the corneal stroma fixed with glutaraldehyde and air-dried, the collagen fibrils show the commonly accepted D-period of 61 ± 8 nm. We used the atomic force microscopy method to image collagen fibrils type I present in the middle layers of human cornea and sclera. The water content in the cornea and sclera samples was varying in the range of .066-.085. Calculations of the D-period using the theoretical model of the fibril and the FFT approach allowed to reveal the possible molecular mechanism of the D-period shortening in the corneal collagen fibrils upon drying. It was found that both the decrease in the shift and the simultaneous reduction in the distance between tropocollagen molecules can be responsible for the experimentally observed effect. We also hypothesize that collagen type V, which co-assembles with collagen type I into heterotypic fibrils in cornea, could be involved in the observed shortening of the corneal D-period.

Synthesis of New Styrylquinoline Cellular Dyes, Fluorescent Properties, Cellular Localization and Cytotoxic Behavior.

New styrylquinoline derivatives with their photophysical constants are described. The synthesis was achieved via Sonogashira coupling using the newly developed heterogeneous nano-Pd/Cu catalyst system, which provides an efficient synthesis of high purity products. The compounds were tested in preliminary fluorescent microscopy studies to in order to identify their preferable cellular localization, which appeared to be in the lipid cellular organelles. The spectroscopic properties of the compounds were measured and theoretical TD-DFT calculations were performed. A biological analysis of the quinolines that were tested consisted of cytotoxicity assays against normal human fibroblasts and colon adenocarcinoma cells. All of the compounds that were studied appeared to be safe and indifferent to cells in a high concentration range. The presented results suggest that the quinoline compounds that were investigated in this study may be valuable structures for development as fluorescent dyes that could have biological applications.

Physicochemical properties of potential porphyrin photosensitizers for photodynamic therapy.

This research evaluated the suitability of synthetic photosensitizers for their use as potential photosensitizers in photodynamic therapy using steady state and time-resolved spectroscopic techniques. Four tetraphenylporphyrin derivatives were studied in ethanol and dimethyl sulfoxide. The spectroscopic properties namely electronic absorption and emission spectra, ability to generate singlet oxygen, lifetimes of the triplet state, as well as their fluorescence quantum yield were determined. Also time-correlated single photon counting method was used to precisely determine fluorescence lifetimes for all four compounds. Tested compounds exhibit high generation of singlet oxygen, low generation of fluorescence and they are chemical stable during irradiation. The studies show that the tested porphyrins satisfy the conditions of a potential drug in terms of physicochemical properties.

Theoretical reproduction of the Q-band absorption spectrum of free-base chlorin.

The computational results of the features observed in the room-temperature Q-band absorption spectrum of free-base chlorin (H2Ch) are presented. The vibrational structures of the first and second excited singlet states were calculated based on a harmonic approximation using density functional theory and its time dependent extension within the Franck-Condon and Herzberg-Teller approaches. The outcome allowed to identify the experimental bands and to assign them to the specific vibrational transitions. A very good agreement between the simulated and measured wavelengths and their relative intensities provided the opportunity to predict the origin of the S0 → S2 transition which could not be determined experimentally.

Exploring the anti-cancer activity of novel thiosemicarbazones generated through the combination of retro-fragments: dissection of critical structure-activity relationships.

Thiosemicarbazones (TSCs) are an interesting class of ligands that show a diverse range of biological activity, including anti-fungal, anti-viral and anti-cancer effects. Our previous studies have demonstrated the potent in vivo anti-tumor activity of novel TSCs and their ability to overcome resistance to clinically used chemotherapeutics. In the current study, 35 novel TSCs of 6 different classes were designed using a combination of retro-fragments that appear in other TSCs. Additionally, di-substitution at the terminal N4 atom, which was previously identified to be critical for potent anti-cancer activity, was preserved through the incorporation of an N4-based piperazine or morpholine ring. The anti-proliferative activity of the novel TSCs were examined in a variety of cancer and normal cell-types. In particular, compounds 1d and 3c demonstrated the greatest promise as anti-cancer agents with potent and selective anti-proliferative activity. Structure-activity relationship studies revealed that the chelators that utilized "soft" donor atoms, such as nitrogen and sulfur, resulted in potent anti-cancer activity. Indeed, the N,N,S donor atom set was crucial for the formation of redox active iron complexes that were able to mediate the oxidation of ascorbate. This further highlights the important role of reactive oxygen species generation in mediating potent anti-cancer activity. Significantly, this study identified the potent and selective anti-cancer activity of 1d and 3c that warrants further examination.

Lessons from chlorophylls: modifications of porphyrinoids towards optimized solar energy conversion.

Practical applications of photosynthesis-inspired processes depend on a thorough understanding of the structures and physiochemical features of pigment molecules such as chlorophylls and bacteriochlorophylls. Consequently, the major structural features of these pigments have been systematically examined as to how they influence the S1 state energy, lifetimes, quantum yields, and pigment photostability. In particular, the effects of the macrocyclic π-electron system, central metal ion (CMI), peripheral substituents, and pigment aggregation, on these critical parameters are discussed. The results obtained confirm that the π-electron system of the chromophore has the greatest influence on the light energy conversion capacity of porphyrinoids. Its modifications lead to changes in molecular symmetry, which determine the energy levels of frontier orbitals and hence affect the S1 state properties. In the case of bacteriochlorophylls aggregation can also strongly decrease the S1 energy. The CMI may be considered as another influential structural feature which only moderately influences the ground-state properties of bacteriochlorophylls but strongly affects the singlet excited-state. An introduction of CMIs heavier than Mg2+ significantly improves pigments' photostabilities, however, at the expense of S1 state lifetime. Modifications of the peripheral substituents may also influence the S1 energy, and pigments' redox potentials, which in turn influence their photostability.

Iron chelators in photodynamic therapy revisited: synergistic effect by novel highly active thiosemicarbazones.

In photodynamic therapy (PDT), a noninvasive anticancer treatment, visible light, is used as a magic bullet selectively destroying cancer cells by a photosensitizer that is nontoxic in the dark. Protoporphyrin IX (PpIX) is a natural photosensitizer synthesized in the cell, which is also a chelating agent that if bonded to Fe(2+) forms heme, a central component of hemoglobin. Therefore, xenobiotic iron chelators can disturb iron homeostasis, increasing the accumulation of PpIX, obstructing the last step of heme biosynthesis, and enhancing PDT efficiency. However, the attempts to use this promising idea have not proved to be hugely successful. Herein, we revisited this issue by analyzing the application of iron chelators highly toxic in the dark, which should have higher Fe(2+) affinity than the nontoxic chelators used so far. We have designed and prepared thiosemicarbazones (TSC) with the highest dark cellular cytotoxicity among TSCs ever reported. We demonstrate that compound 2 exerts powerful PDT enhancement when used in combination with 5-aminolevulinic acid (ALA), a precursor of PpIX.

Theoretical investigation of porphyrin-based photosensitizers with enhanced NIR absorption.

Porphyrins are compounds which are fundamental for designing many photosensitizers assigned for use in photodynamic therapy (PDT). However, photosensitizers available on the drug market are not ideal for use in PDT among others because of their low absorption in the wavelength range optimal for tissue penetration, i.e. the near-infrared (NIR) spectral region. In the present study the density functional theory and its time dependent extension calculations have been used to design new porphyrin-based photosensitizers with absorption in the therapeutic window of PDT of 700-800 nm. Theoretical investigation of four different derivatives of the 5,10,15,20-tetrakis(4-hydroxyphenyl)porphyrin (p-THPP) revealed specific 'bridge' configurations which have a significant influence on absorption spectra. The results of the present study may be a useful starting point for future design of porphyrin derivatives as improved photosensitizers.

DFT/TD-DFT study of solvent effect as well the substituents influence on the different features of TPP derivatives for PDT application.

Spectral characteristics study of meso-tetraphenylporphyrin derivatives (TPP1 and TPP2) used as photosensitizers for utilization in photodynamic therapy (PDT) has been performed by density functional theory (DFT) and time dependent DFT (TD-DFT) calculations at B3LYP/6-31 G(d) level of theory using PCM solvation model. The geometrical parameters of porphyrins have been studied for ground and excited-state geometry to deduce the influence of various substituents as well as solvent effect on the deformation of porphyrin ring. Two theoretical approaches - linear response (LR) and external iteration (EI) - have been performed to replicate absorption and fluorescence emission spectra. Experimental and theoretical investigations have shown that EI method reproduces the absorption energies very well for both singlet-singlet and triplet-triplet transitions, whereas the LR approach is more coherent with experimental fluorescence emission spectra. Spectral features and HOMO-LUMO band gap analysis have shown that TPP1 can be more useful in PDT. Calculations have revealed that two the highest occupied and two the lowest unoccupied molecular orbitals are responsible for the Q-band absorption and are located mainly on the porphyrin ring. In order to verify the substituent effect on the activity of tested compounds in their ground and excited states, the molecular electrostatic potential surfaces have been analyzed.

Investigation of the biological properties of (hetero)aromatic thiosemicarbazones.

Two series of thiosemicarbazone-based iron chelators (twenty-seven compounds) were designed and synthesized using a microwave-assisted approach. Quinoline and halogenated phenyl were selected as parent scaffolds on the basis of a similarity search. The lipophilicity of the synthesized compounds was measured using HPLC and then calculated. Primary in vitro screening of the synthesized compounds was performed against eight pathogenic fungal strains. Only a few compounds showed moderate activity against fungi, and (E)-2-(quinolin-2-ylvinyl)-N,N-dimethylhydrazine-carbothioamide appeared to be more effective than fluconazole against most of the fungal strains tested. Antiproliferative activity was measured using a human colon cancer cell line (HCT-116). Several of the tested compounds showed submicromolar antiproliferative activity. Compounds were also tested for their activity related to the inhibition of photosynthetic electron transport (PET) in spinach (Spinacia oleracea L.) chloroplasts. The structure-activity relationships are discussed for all of the compounds.

meso-5,10,15,20-Tetra-kis(4-hy-droxy-3-meth-oxy-phen-yl)porphyrin propionic acid monosolvate.

In the title compound, C(48)H(38)N(4)O(8)·C(3)H(6)O(2), the porphyrin mol-ecule is centrosymmetric. The propionic acid solvent mol-ecule is disordered over two sets of sites with equal occupancy factors. The porphyrin central core is almost planar, with an r.m.s. deviation of the fitted atoms of 0.045 Å. The substituent benzene rings make dihedral angles of 70.37 (4) and 66.95 (4)° with respect to the porphyrin core plane. The crystal structure is stabilized by an inter-esting network of hydrogen bonds. Porphyrin mol-ecules are connected by O-H⋯O hydrogen bonds creating ribbons running along the [101] direction. Weak C-H⋯O hydrogen bonds connect separate mol-ecular ribbons in the [110] direction, creating (-111) layers. Intra-molecular N-H⋯N hydrogen bonds also occur. The propionic acid molecules are connected by pairs of -H⋯O hydrogen bonds, creating dimers.

Synthesis and characterization of quinoline-based thiosemicarbazones and correlation of cellular iron-binding efficacy to anti-tumor efficacy.

Iron chelators have emerged as a potential anti-cancer treatment strategy. In this study, a series of novel thiosemicarbazone iron chelators containing a quinoline scaffold were synthesized and characterized. A number of analogs show markedly greater anti-cancer activity than the 'gold-standard' iron chelator, desferrioxamine. The anti-proliferative activity and iron chelation efficacy of several of these ligands (especially compound 1b), indicates that further investigation of this class of thiosemicarbazones is worthwhile.

Investigating the anti-proliferative activity of styrylazanaphthalenes and azanaphthalenediones.

A group of styrylazanaphthalenes and azanaphthalenediones were synthesized and tested for their anti-proliferative activity. Most of the compounds were obtained with the use of microwave-assisted synthesis. The lipophilicity of the compounds was measured by RP-HPLC and their anti-proliferative activity was assayed against the human SK-N-MC neuroepithelioma and HCT116 human colon carcinoma cell lines. Active compounds were also tested in clonogenity and comet assays. Several quinazolinone and styrylquinazoline analogues were found to have markedly greater anti-proliferative activity than desferoxamine and cis-platin.

Spectroscopic and biological studies of a novel synthetic chlorin derivative with prospects for use in PDT.

Photosensitizers with desirable combinations of chemical, photophysical and biological properties are essential for improving the efficacy of photodynamic therapy (PDT) against various cancers. Chlorins seem to be promising candidates for photodynamic therapy (PDT) owing to their photophysical properties. This paper reports spectroscopic and biological properties of a novel synthetic chlorin derivative. Cytotoxicity, phototoxicity as well as subcellular localization of the novel derivative was studied using Lewis lung carcinoma cultured cells (LLC). In the examined concentration range no significant cytotoxic effects were found but high phototoxicity was observed. Confocal laser scanning microscopy demonstrated that the compound, upon entering cells, was localized in the perinuclear cytoplasm of LLC cells. Using fluorescent microscopy we investigated the impact of PDT based on the novel compound upon cytoskeleton and DNA structure of LLC cells. Our results indicate that liposomes are effective in transferring the chlorin photosensitizer into the studied cells, leading to their high photosensitization, whereas the non-carrier delivery mode (i.e., DMSO) is rather useless for such purposes.

N,N'-Bis(2-hydroxy-ethyl)benzene-1,4-dicarboxamide.

The mol-ecule of the title compound, C(12)H(16)N(2)O(4), is centrosymmetric and the amide group is twisted relative to the benzene ring by 14.40 (13)°. The mol-ecules are hydrogen bonded into a three-dimensional framework, with the hydr-oxy O atoms acting as acceptors in N-H⋯O hydrogen bonds and as donors in O-H⋯O=C inter-actions.

Spectroscopic properties and photodynamic effects of new lipophilic porphyrin derivatives: efficacy, localisation and cell death pathways.

Photodynamic therapy (PDT) and photodynamic diagnostics (PDD) of cancer are based on the use of non-toxic dyes (photosensitisers) in combination with harmless visible light. This paper reports physicochemical properties, cell uptake, localisation as well as photodynamic efficiency of two novel lipophilic porphyrin derivatives, suitable for use as PDT sensitisers. Both compounds are characterised by high quantum yield of singlet oxygen generation which was measured by time-resolved phosphorescence. Photodynamic in vitro studies were conducted on three cancer cell lines. Results of cell survival tests showed negligible dark cytotoxicity but high phototoxicity. The results also indicate that cell death is dependent on energy dose and time following light exposure. Using confocal laser scanning microscopy both compounds were found to localise in the cytoplasm around the nucleus of the tumour cells. The mode of cell death was evaluated based on the morphological changes after differential staining. In summary, good photostability, high quantum yield of singlet oxygen and biological effectiveness indicate that the examined lipophilic porphyrin derivatives offer quite interesting prospects of photodynamic therapy application.