phytochlorin and pheophorbide-a

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

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 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

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