chlorophyll-a and phytochlorin

To evaluate the effect of drug hydrophobicity on nanoparticle delivery in vivo, we conducted a comparative study using different photosensitizer-loaded poly(lactic-co-glycolic acid) (PLGA) nanoparticles (NPs). Chlorin e6 (Ce6) and pheophorbide a (Pba) with similar structure but different hydrophobicity were loaded into PLGA-NPs separately. We observed release profiles and photodynamic effects in vitro from the resulting Ce6- and Pba-PLGA-NPs. After intravenous injection into SCC7 tumor-bearing mice, biodistribution and accumulation of two drugs in tumor tissue were observed by real-time fluorescence imaging. Finally, in vivo photodynamic therapy with Ce6- and Pba-PLGA-NPs provided different therapeutic results according to imaging data. The results demonstrated that drug hydrophobicity is an important factor in nanoparticle drug delivery and should be considered for efficient drug delivery in vivo.

Topics: Animals; Cell Line, Tumor; Cell Survival; Chlorophyll; Chlorophyllides; Drug Delivery Systems; Flow Cytometry; Hydrophobic and Hydrophilic Interactions; Mice; Nanoparticles; Photochemotherapy; Photosensitizing Agents; Polylactic Acid-Polyglycolic Acid Copolymer; Porphyrins

Tailor-made drug solubilizers are studied based on peptide-poly(ethylene glycol) conjugates, which exhibit peptide segments constituting binding motifs for the small-molecule drugs of interest to render them water-soluble. Suitable 7mer peptides are selected via combinatorial means by screening large one-bead-one-compound (OBOC) peptide libraries. The capability of the screening method to read out structural detail of the drugs is investigated by comparing three related photosensitizers (Chlorin E6 (Ce6), Pheophorbide A (Pba) and meta-tetra(hydroxyphenyl)chlorin (m-THPC), which are applicable for photodynamic cancer therapy. The screening procedure delivers de novo solubilizers that show the best solubilization efficiency for the drug the screening is performed with. While molecular recognition events between peptide and drug are not expected to be found, significant binding capacity differences of, e.g., the Ce6-solubilizer for Pba are suggesting selectivity in drug binding, even among structurally closely related drugs. Cyro-Electron microscopy revealed the formation of colloidal aggregates between drug moieties and peptide conjugates. Insights into relevant amino acids in the identified peptide sequences are gained by studying capacities of systematic point mutations (alanine scans), enabling understanding of drug-binding motifs. These reveal the importance of sequence positioning of appropriate H-bonding between polar functional groups of the peptide and the drugs, which agrees well with computational binding studies performed on drug/peptide model complexes.

Topics: Amino Acid Sequence; Chlorophyll; Chlorophyllides; Mesoporphyrins; Models, Molecular; Peptides; Photochemotherapy; Photosensitizing Agents; Polyethylene Glycols; Porphyrins; Solubility

In the present work, we investigated the dark and photoinduced cytotoxic activity of the new chlorophyll-a derivatives which contain the substituents of oligoethylene glycol on the periphery of their macrocycles. These compounds were tested using human cell lines to estimate their potential as photosensitizers for photodynamic therapy of cancer. It was shown that all the tested compounds have expressed photoinduced cytotoxic activity in vitro. Detailed study of the biological activity of one of the most perspective compound in this series-pyropheophorbide-a 17-diethylene glycol ester (Compound

Topics: A549 Cells; Animals; Apoptosis; Cell Line; Cell Survival; Chlorophyll; Chlorophyllides; Comet Assay; Darkness; DNA Damage; Dose-Response Relationship, Drug; Gene Expression; HEK293 Cells; HeLa Cells; Hemolysis; Humans; Inhibitory Concentration 50; Light; Mice; Microscopy, Fluorescence; Molecular Structure; Photosensitizing Agents; Polyethylene Glycols; Porphyrins

Quantification of fluorescence in vivo is complicated by the influence of tissue optical properties on the collected fluorescence signal. When tissue optical properties in the measurement volume are quantified, one can obtain the intrinsic fluorescence, which equals the product of fluorophore absorption coefficient and quantum yield. We applied this method to in vivo single-fiber fluorescence spectroscopy measurements on mouse tongue, skin, liver, and oral squamous cell carcinoma, where we detected intrinsic fluorescence spectra of the photosensitizers chlorin e6 and Bremachlorin at t=[3,4.5,6,24,48]  h incubation time. We observed a tissue-dependent maximum of 35% variation in the total correction factor over the visible wavelength range. Significant differences in spectral shape over time between sensitizers were observed. Although the wavelength position of the fluorescence intensity maximum for ce6 shifted to the red, Bremachlorin showed a blue shift. Furthermore, the Bremachlorin peak appeared to be broader than the ce6 fluorescence peak. Intrinsic fluorescence intensity, which can be related to photosensitizer concentration, was decreasing for all time points but showed significantly more Bremachlorin present compared to ce6 at long incubation times. Results from this study can be used to define an optimal treatment protocol for Bremachlorin-based photodynamic therapy.

Topics: Animals; Carcinoma, Squamous Cell; Chlorophyll; Chlorophyllides; Female; Fluorescence; Green Fluorescent Proteins; Liver; Mice; Mice, Inbred BALB C; Mice, Nude; Microscopy, Fluorescence; Mouth Neoplasms; Normal Distribution; Optics and Photonics; Photochemotherapy; Photosensitizing Agents; Porphyrins; Skin; Spectrometry, Fluorescence; Spectrophotometry; Tongue

Chlorin-e6 (chl-e6) and a hydrogenated derivative (chl-e6H) were semi-synthesized, and their photophysical properties and photodynamic activity against Escherichia coli, Staphylococcus aureus and Candida albicans evaluated. Methyl pheophorbide-a (Mepheo-a) was obtained from S. maxima using methanolic extraction with acid catalysis (CH3OH–H2SO4). Chlorin-e6 was prepared from Mepheo-a by basic hydrolysis with H2O–acetone and NaOH. Hydrogenated Chlorin-e6 was synthesized by a similar procedure starting from the hydrogenated methyl pheophorbide-a (Mepheo-aH). Photophysical studies were performed in order to determine the singlet oxygen quantum yield of chl-e6H which is higher than that of chl-e6. The microorganism inactivation of chl-e6 and chl-e6H was investigated at two concentrations and three fluence levels. Both chl-e6 and chl-e6H showed microorganism inactivation against Gram-positive bacteria and a fungus.

Topics: Anti-Infective Agents; Candida albicans; Chlorophyll; Chlorophyll A; Chlorophyllides; Escherichia coli; Hydrogenation; Molecular Structure; Photobleaching; Photochemical Processes; Photochemotherapy; Photosensitizing Agents; Porphyrins; Staphylococcus aureus

Solar fuels, such as hydrogen gas produced from water and methanol produced from carbon dioxide reduction by artificial photosynthesis, have received considerable attention. In natural leaves the photosynthetic proteins are well-organized in the thylakoid membrane. To develop an artificial leaf device for solar low-carbon fuel production from CO2, a chlorophyll derivative chlorin-e6 (Chl-e6; photosensitizer), 1-carboxylundecanoyl-1'-methyl-4,4'-bipyrizinium bromide, iodide (CH3V(CH2)9COOH; the electron carrier) and formate dehydrogenase (FDH) (the catalyst) immobilised onto a silica-gel-based thin layer chromatography plate (the Chl-V-FDH device) was investigated. From luminescence spectroscopy measurements, the photoexcited triplet state of Chl-e6 was quenched by the CH3V(CH2)9COOH moiety on the device, indicating the photoinduced electron transfer from the photoexcited triplet state of Chl-e6 to the CH3V(CH2)9COOH moiety. When the CO2-saturated sample solution containing NADPH (the electron donor) was flowed onto the Chl-V-FDH device under visible light irradiation, the formic acid concentration increased with increasing irradiation time.

Topics: Bioelectric Energy Sources; Biofuels; Biomimetic Materials; Carbon Dioxide; Chlorophyll; Chlorophyllides; Electron Transport; Formate Dehydrogenases; Formates; Green Chemistry Technology; Hydrogen; Light; NADP; Oxidation-Reduction; Photochemistry; Photosensitizing Agents; Photosynthesis; Plant Leaves; Porphyrins; Silica Gel; Solar Energy; Thylakoids

Disinfection of the tooth pulp-canal system is imperative to successful endodontic therapy. Yet, studies suggest that 30-50% of current endodontic treatments fail from residual bacterial infection. Photodynamic therapy using red-light chromophores (630 nm) to induce antimicrobial death mediated by generated reactive oxygen species (ROS) has been reported, but red-light also may thermally damage resident tissues. In the current study, we tested the hypothesis that several blue light chromophores (380-500 nm) generate intracellular reactive oxygen species but are not cytotoxic to mammalian cells.. THP1 monocytes were exposed to 10 microM of four chromophores (chlorin e6, pheophorbide-a, pheophorbide-a-PLL, and riboflavin) for 30 min before activation with blue light (27J/cm(2), 60s). After activation, intracellular ROS were measured using a dihydrofluorescein diacetate technique, and cytotoxicity was determined by measuring mitochondrial activity with the MTT method.. All photosensitizers produced intracellular ROS levels that were dependent on both the presence of the photosensitizer and blue light exposure. Riboflavin and pheophorbide-a-PLL produced the highest levels of ROS. Photosensitizers except riboflavin exhibited cytotoxicity above 10 microM, and all except pheophorbide-a-PLL were more cytotoxic after blue light irradiation.. The current study demonstrated the possible utility of blue light chromophores as producers of ROS that would be useful for endodontic disinfection.

Topics: Cell Line; Chlorophyll; Chlorophyllides; Coloring Agents; Fluoresceins; Fluorescent Dyes; Humans; Light; Mitochondria; Monocytes; Photosensitizing Agents; Polylysine; Porphyrins; Radiation-Sensitizing Agents; Reactive Oxygen Species; Riboflavin; Succinate Dehydrogenase; Tetrazolium Salts; Thiazoles

The two photosensitizers, chlorin e6 and pheophorbide a, were tested in an in vitro model of topical photodynamic therapy (PDT). Both dyes accumulate in HaCaT keratinocytes as verified by fluorescence measurement but pheophorbide a is enriched fivefold more strongly than chlorin e6 after 24 h. HaCaT cells are susceptible to PDT with both dyes. The phototoxicity measured by ATP bioluminescence is caused by necrosis and apoptosis depending on the photosensitizer used and the treatment modality. Chlorin e6 shows higher toxic potential because it elicits nearly 90% cell mortality 24 h after PDT comparable to pheophorbide a but with a fivefold lower rate of accumulation. These results implicate caution with topical PDT of oncologic diseases due to the risk of serious side effects on healthy skin in the course of topical photodynamic treatment. But the lack of dark toxicity and the time-dependent enrichment of both dyes in HaCaT cells are arguments for the application of these sensitizers in topical PDT of non-malign skin disorders. Further studies are necessary to discover appropriate lower doses and mechanisms of action of topical PDT with both compounds.

Topics: Apoptosis; Caspase 3; Cells, Cultured; Chlorophyll; Chlorophyllides; Humans; Keratinocytes; Photochemotherapy; Porphyrins; Structure-Activity Relationship

The primary event in vision is light-initiated activation of visual pigments. All visual pigments consist of the protein opsin bound to 11-cis-retinal and are responsible for initiating the transformation of light into an electrical signal. In a mouse model, we show that derivatives of chlorophyll can act as visual pigments initiating the transformation of light into an electrical signal and thus change the primary event in vision to initial activation of a chlorophyll derivative. Electroretinographic b-wave amplitudes recorded in response to red and blue light were two-fold greater in mice administered chlorin e(6), which accumulated in photoreceptor outer segments.

Topics: Animals; Chlorophyll; Chlorophyllides; Color Perception; Light; Mice; Mice, Inbred BALB C; Molecular Structure; Photic Stimulation; Porphyrins; Retina; Retinal Pigments; Vision, Ocular

Three cationic water-soluble chlorin e(6) derivatives, that is, 6a-,gammab-,7c-tris(2-trimethylammonioethyl)chlorin e(6) (1), 6a-,gammab-,7c-tris(3-methylpyridiniummethyl)chlorin e(6) (2), and 6a-,gammab-, 7c-tris(2-trimethylammonioethyl)-2-(3-trimethylammonioprop-1-enyl)chlorin e(6) (3), have been designed and synthesized to allow the study of their DNA-binding and -photocleavage activities. The DNA-unwinding assay, measurements of melting temperatures of double-stranded DNA, and the induced CD and visible absorption spectra have revealed that 1 and 3 are intercalated into the base pairs of the double-helical DNA, while 2 is bound to outside the minor groove of the double-helical DNA. The cationic water-soluble chlorin e(6) derivatives effectively cleave the double-helical DNA under photoirradiation and the DNA-photocleavage activity increases in the order 3>1>2. The DNA-binding and -photocleavage characteristics of the three cationic water-soluble chlorin e(6) derivatives are influenced by aspects of their molecular structure, such as the kind, number, and position of the cationic substituents.

Topics: Cations; Chemical Phenomena; Chemistry, Physical; Chlorophyll; Chlorophyllides; Circular Dichroism; DNA; Photochemistry; Porphyrins; Radiation-Sensitizing Agents; Solubility; Water

In the absence of a red-sensitive visual pigment, some deep-sea fish use a chlorophyll derivative in their green-sensitive rod cells in order to see deep-red light. Here we show that living rods extracted from a salamander can also accumulate an exogenous chlorophyll derivative, chlorin e6, that renders them as sensitive to red light as they are to green. This vision enhancement by an unbleachable chlorophyll derivative might therefore be a general phenomenon in vertebrate photoreception.

Topics: Animals; Cattle; Chlorophyll; Chlorophyllides; Color; Color Perception; Porphyrins; Retinal Rod Photoreceptor Cells; Rhodopsin; Urodela

The Qy absorption band of two chlorophyll derivatives, zinc chlorin e6 (ZnCe6) and zinc pheophorbide a (ZnPheida), in aqueous solution is bathochromically shifted on addition of quinones, e.g., 1,4-benzoquinone (BQ), with a corresponding shift of the fluorescence band. This is due to a complex formation of zinc chlorins induced by BQs and subsequent rearrangement. The time-resolved absorption spectra after laser pulse excitation show triplet quenching of the pigments by BQ and other quinones via electron transfer. The effects of electron transfer to noncovalently bound BQs were also studied with de novo synthesized peptides, into which ZnCe6 and ZnPheida were incorporated as model systems for the primary steps of photosynthetic reaction centers. Whereas the photophysical properties are similar to those of the unbound zinc chlorins, no BQ-mediated complex formation was observed.

Topics: Benzoquinones; Chlorophyll; Chlorophyllides; Kinetics; Organometallic Compounds; Porphyrins; Protein Conformation; Spectrometry, Fluorescence; Spectrophotometry, Ultraviolet; Zinc

In photodynamic therapy (PDT), a tumor-selective photosensitizer is administered followed by activation of the photosensitizer by exposure to a light source of a given wavelength. This, in turn, generates reactive oxygen species that induce cellular apoptosis and necrosis in tumor tissue. Based on our earlier finding that the photosensitizer pheophorbide a is an ABCG2 substrate, we explored the ability of ABCG2 to transport photosensitizers with a structure similar to that of pheophorbide a. ABCG2-overexpressing NCI-H1650 MX50 bronchoalveolar carcinoma cells were found to have reduced intracellular accumulation of pyropheophorbide a methyl ester and chlorin e6 compared to parental cells as measured by flow cytometry. The ABCG2 inhibitor fumitremorgin C was found to abrogate ABCG2-mediated transport. Intracellular fluorescence of hematoporphyrin IX, meso-tetra(3-hydroxyphenyl)porphyrin, and meso-tetra(3-hydroxyphenyl)chlorin was not substantially affected by ABCG2. ABCG2-overexpressing cells also displayed decreased intracellular fluorescence of protoporphyrin IX generated by exogenous application of 5-aminolevulinic acid. Mutations at amino acid 482 in the ABCG2 protein known to affect substrate specificity were not found to impact transport of the photosensitizers. In cytotoxicity assays, ABCG2-transfected HEK-293 cells were 11-fold, 30-fold, 4-fold, and >7-fold resistant to PDT with pheophorbide a, pyropheophorbide a methyl ester, chlorin e6, and 5-aminolevulinic acid, respectively. ABCG2-transfected cells were not resistant to PDT with meso-tetra(3-hydroxyphenyl) chlorin. Neither multidrug resistance-associated protein 1 expression nor P-glycoprotein expression appreciably decreased the intracellular fluorescence of any of the photosensitizers examined as determined by flow cytometry. The results presented here implicate ABCG2 as a possible cause for cellular resistance to photodynamic therapy.

Topics: Adenocarcinoma, Bronchiolo-Alveolar; Aminolevulinic Acid; ATP Binding Cassette Transporter, Subfamily B, Member 1; ATP Binding Cassette Transporter, Subfamily G, Member 2; ATP-Binding Cassette Transporters; Biological Transport; Cell Proliferation; Chlorophyll; Chlorophyllides; Flow Cytometry; Fluorescence; Humans; Indoles; Kidney; Lung Neoplasms; Multidrug Resistance-Associated Proteins; Neoplasm Proteins; Photochemotherapy; Photosensitizing Agents; Porphyrins; Protoporphyrins; Tumor Cells, Cultured

A bio-photovoltaic conversion device based on dye-sensitised solar cell (DSSC) using the visible light sensitisation of chlorine-e6 (Chl-e6) derived from chlorophyll from Spirulina adsorbed on a nanocrystalline TiO2 film was developed. Form fluorescence spectrum of Chl-e6 adsorbed on a nanocrystalline TiO2 film, the emission of Chl-e6 was effectively quenched by TiO2 nanocrystalline indicating that the effective electron injection from the excited singlet state of Chl-e6 into the conduction band of TiO2 particles occurred. The short-circuit photocurrent density (Isc). the open-circuit photovoltage (Voc). and the fill factor (FF) of solar cell using Chl-e6 adsorbed on a nanocrystalline TiO2 film electrode were estimated to be 0.305 +/- 0.012 mA cm(-2), 426 +/- 10 mV, and 45.0%, respectively. IPCE values were reached a maximum around the wavelength of absorption maximum (7.40% at 400 nm; 1.44% at 514 nm and 2.91% at 670 nm), indicating that the DSSC using visible light sensitisation of nanocrystalline TiO2 film by Chl-e6 was developed.

Topics: Bacterial Proteins; Chlorophyll; Chlorophyllides; Crystallization; Electric Power Supplies; Electrochemistry; Electrodes; Equipment Design; Equipment Failure Analysis; Light; Nanotechnology; Photochemistry; Porphyrins; Reproducibility of Results; Sensitivity and Specificity; Spirulina; Titanium; Transducers

Amphiphilic sensitizers self-associate in aqueous environments and form aggregated species that exhibit no or only negligible photodynamic activity. However, amphiphilic photosensitizers number among the most potent agents of photodynamic therapy. The processes by which these sensitizers are internalized into tumor cells have yet to be fully elucidated and thus remain the subject of debate. In this study the uptake of photosensitizer aggregates into tumor cells was examined directly using subcellular time-resolved fluorescence spectroscopy with a high temporal resolution (20-30 ps) and high sensitivity (time-correlated single-photon counting). The investigations were performed on selected sensitizers that exhibit short fluorescence decay times (< 50 ps) in aggregated form. Derivatives of pyropheophorbide-a ether and chlorin e6 with varying lipophilicity were used for the study. The characteristic fluorescence decay times and spectroscopic features of the sensitizer aggregates measured in aqueous solution also could be observed in A431 human endothelial carcinoma cells administered with these photosensitizers. This shows that tumor cells can internalize sensitizers in aggregated form. Uptake of aggregates and their monomerization inside cells were demonstrated directly for the first time by means of fluorescence lifetime imaging with a high temporal resolution. Internalization of the aggregates seems to be endocytosis mediated. The degree of their monomerization in tumor cells is strongly influenced by the lipophilicity of the compounds.

Topics: Chlorophyll; Chlorophyllides; Fluorescence; Humans; Kinetics; Light; Molecular Structure; Photons; Photosensitizing Agents; Porphyrins; Solutions; Spectrometry, Fluorescence; Time Factors; Tumor Cells, Cultured

A methanolic crude extract of the plant Garuga pinnata Roxb. (Burseraceae) showed promising cytotoxic activity against a panel of human tumor cell lines in vitro, including KB and its drug-resistant sublines (Ferguson et al. Cancer Res. 1988, 48, 5956). Pheophorbide-a and-b methyl esters (3,4) were isolated as active principles with broad photo-dependent cytotoxic activities in the micromolar range. These findings prompted SAR studies of known and novel pheophorbide-a derivatives as photo-dependent and photo-independent cytotoxic agents. The results showed that zinc-protoporphyrin IX (10), zinc 13(R)-hydroxypheophorbide-a methyl ester (22), and zinc chlorin-e6 trimethyl ester (13) possessed photo-independent cytotoxic activity. Compounds 13 and 22 were the most active cytotoxic agents of the series (mean ED(50) 4.6 +/- 1.0 microM and 5.7 +/- 0.7 microM, respectively) against KB cells incubated in the dark.

Topics: Antineoplastic Agents; Cell Division; Chlorophyll; Chlorophyllides; DNA Damage; Drug Screening Assays, Antitumor; Humans; Light; Porphyrins; Protoporphyrins; Radiation-Sensitizing Agents; Structure-Activity Relationship; Tumor Cells, Cultured; Zinc

We propose the use of acetoxymethyl esters of pH-sensitive amphipathic photosensitizers (PS) for photodynamic therapy (PDT). These compounds may be applicable for PDT involving endocytosis of lipophilic carriers leading to lysosomal uptake of the esterified PS by target cells. Partial and/or total enzymatic de-esterification may result in the extralysosomal distribution of the photoactive agents, possibly culminating in a multisite photochemical response. We report here the synthesis and properties of chlorin e6 triacetoxymethyl ester (CAME) and pheophorbide a acetoxymethyl ester (PAME). Chlorin e6 and pheophorbide a are photocytotoxic chlorins that possess free carboxylate groups and exhibit optimum wavelengths of excitation substantially red shifted relative to hematoporphyrin derivative. Acetoxymethyl esterification of chlorin e6 and pheophorbide a was accomplished with bromomethyl acetate. High-performance liquid chromatography allowed for the purification of PAME, in 87% purity, and CAME, in 63% yield and 94% purity, as well as the detection of the presumed mono- and diesters of chlorin e6 as transient intermediates in the synthesis of CAME. The ultraviolet-visible absorption, fluorescence excitation and emission, NMR and mass spectra of the chlorin e6 triester are consistent with those expected for CAME. The pH-sensitive amphipathicity of pheophorbide a and chlorin e6 but not CAME was demonstrated using a water/1-octanol partition assay. The production of pheophorbide a from PAME and the sequential formation of the di- and monoesters and free chlorin e6 from CAME, by the action of lysosomal esterases obtained from cancer cells, demonstrate the potential of cellular enzymes to convert the lipophilic esters to pH-sensitive amphipathic PS.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Carcinoma; Chlorophyll; Chlorophyllides; Esterases; Esterification; Esters; Humans; Hydrogen-Ion Concentration; Lysosomes; Photosensitizing Agents; Porphyrins; Solubility; Spectrometry, Fluorescence; Spectrophotometry; Urinary Bladder Neoplasms