Chlorin e6 131:152-Anhydride: A Key Intermediate in Conjugation Reactions of Chlorin e6.
ABSTRACT: Since the patent for the photodynamic therapy agent Talaporfin (mono-L-aspartylchlorin e6) was issued in 1987, confusion has existed regarding which of the three carboxylic acid groups in the chlorophyll degradation product, chlorin e6 (1), is modified in standard amino acid type conjugations (using DCC or EDC and an organic base) with amino acids and other biomolecules. Here it is shown that the site of conjugation is the central 152-carboxylic acid, such reactions proceeding in numerous examples via a 131,152-anhydride for which a high resolution X-ray structure is reported. Conjugation with eight oxygen and nitrogen nucleophiles, in every case, afforded the 152-conjugate, reinforcing the earlier conclusion that Talaporfin is the 152-aspartyl conjugate of chlorin e6 and suggesting that reports of 173-conjugation of chlorin e6 using stoichiometric peptide coupling procedures should be subjected to further scrutiny.
Project description:M2-like tumor-associated macrophages (M2-TAMs) in cancer tissues are intimately involved in cancer immunosuppression in addition to growth, invasion, angiogenesis, and metastasis. Hence, considerable attention has been focused on cancer immunotherapies targeting M2-TAMs. However, systemic therapies inhibit TAMs as well as other macrophages important for normal immune responses throughout the body. To stimulate tumor immunity with fewer side effects, we targeted M2-TAMs using photodynamic therapy (PDT), which damages cells via a nontoxic photosensitizer with harmless laser irradiation. We synthesized a light-sensitive compound, mannose-conjugated chlorin e6 (M-chlorin e6), which targets mannose receptors highly expressed on M2-TAMs. M-chlorin e6 accumulated more in tumor tissue than normal skin tissue of syngeneic model mice and was more rapidly excreted than the second-generation photosensitizer talaporfin sodium. Furthermore, M-chlorin e6 PDT significantly reduced the volume and weight of tumor tissue. Flow cytometric analysis revealed that M-chlorin e6 PDT decreased the proportion of M2-TAMs and increased that of anti-tumor macrophages, M1-like TAMs. M-chlorin e6 PDT also directly damaged and killed cancer cells in vitro. Our data indicate that M-chlorin e6 is a promising new therapeutic agent for cancer PDT.
Project description:Syntheses of three new chlorin e6 conjugates for PDT of tumors are reported. One of the new compounds 17 is conjugated with lysine at the 131-position, but the others are mono-conjugated 14 or diconjugated 15 with the non-amino acid species ethanolamine. Cellular experiments with the three new compounds and previously synthesized non-amino acid 152-conjugates (7-10), 131-monoconjugates 14, 16, and a 131,152-diconjugate 12 are reported. In vitro cytotoxicity experiments show that the 131-conjugates are more toxic than the 152-conjugates, and the most toxic derivative (dark- and photo-toxicity) is the 131-ethylenediamine conjugate 11. The most useful PDT photosentitizers appear to be the ethanolamine derivatives, conjugated at the 152- and the 131,152-positions; these show high phototoxicity but relatively low dark toxicity compared with 11, and also the highest dark/photo cytotoxicity ratios.
Project description:Chlorin e6 is a tricarboxylic acid degradation product of chlorophyll a. Four chlorin e6 bis(amino acid) conjugates were regioselectively synthesized bearing two aspartate conjugates in the 13(1),17(3)- and 15(2),17(3)-positions, or at the 13(1),15(2)via an ethylene diamine linker. One additional conjugate bearing two different amino acids, lysine at 13(1)via an ethylene diamine linker and an aspartate at 15(2)via a ?-alanine linker was also synthesized. The cytotoxicity and uptake of four di(amino acid) chlorin e6 conjugates were investigated in human HEp2 cells, and compared with chlorin e6. The most cytotoxic and most taken up conjugates were the zwitterionic 13(1),15(2)-disubstituted conjugates 28 and 33; these also localized in multiple organelles. In contrast, the anionic 13(1),17(3)- and 15(2),17(3)-di-aspartyl chlorin e6 conjugates 12 and 13 showed low dark cytoxicity and lower phototoxicity compared with chlorin e6.
Project description:The self-assembly of A? to ?-sheet-rich neurotoxic oligomers is a main pathological event leading to Alzheimer's disease (AD). Selective targeting of A? oligomers without affecting other functional proteins is therefore an attractive approach to prevent the disease and its progression. In this study, we report that photodynamic treatment of A? in the presence of catalytic amounts of chlorin e6 can selectively damage A? and inhibit its aggregation and toxicity. Chlorin e6 also reversed the amyloid aggregation process in the dark by binding its soluble and low molecular weight oligomers, as shown by thioflavin T (ThT) fluorescence and photoinduced cross-linking of unmodified protein (PICUP) methods. Using HSQC NMR spectroscopy, ThT assays, amino acid analysis, SDS/PAGE, and EPR spectroscopy, we show that catalytic amounts of photoexcited chlorin e6 selectively damage the A? histidine residues H6, H13, and H14, and induce A? cross-linking by generating singlet oxygen. In contrast, photoexcited chlorin e6 was unable to cross-link ubiquitin and ?-synuclein, demonstrating its high selectivity for A?. By binding to the A? histidine residues, catalytic amounts of chlorin e6 can also inhibit the Cu2+-induced aggregation and toxicity in darkness, while at stoichiometric amounts it acts as a chelator to reduce the amount of free Cu2+. This study demonstrates the great potential of chlorin e6 as a multifunctional agent for treatment of AD, and shows that the three N-terminal A? histidine residues are a suitable target for A?-specific drugs.
Project description:In photodynamic therapy (PDT), chlorin e6 (Ce6), with its high phototoxic potential and strong absorption of visible light, penetrates deeply into photodamaged tissue. However, despite this fact, the direct application of Ce6 to PDT has been limited by its low water solubility and poor cancer cell localization. To ameliorate this situation, we report herein on the use of a hydrophilic nanoconjugate (DC) comprised of Ce6 and poly(amidoamine) dendrimer, which improves the water solubility and intracellular uptake of Ce6, thereby enhancing PDT efficacy. The synthesis of DC was verified by ¹H nuclear magnetic resonance (NMR) analysis, and the coupling ratio of Ce6 introduced onto DC was 2.64. The prepared DC was spherical, with an average diameter of 61.7 ± 3.5 nm. In addition, the characteristic ultraviolet-visible absorption bands of DC in distilled water were similar to those of free Ce6 in dimethyl sulfoxide (DMSO), indicating that the Ce6 chromophore did not change upon conjugation. Investigation using fluorescence spectroscopy and confocal microscopy revealed a greater intracellular uptake of DC than of Ce6 alone. Moreover, DC exhibited significantly increased phototoxicity to human cervical cancer cells, mostly because of apoptotic cell death. These results imply that DC is a candidate for the clinical treatment of PDT.
Project description:PEGylated chlorin e(6) photosensitizers were synthesized with tri(ethylene glycol) attached at the ester bond(s) for a 1:1 conjugate at the 17(3)-position, a 2:1 conjugate at the 15(2)- and 17(3)-positions, and a 3:1 conjugate at the 13(1)-, 15(2)-, and 17(3)-positions. These chlorin sensitizers were studied for hydrolytic stability and solubility, as well as ovarian OVCAR-5 cancer cell uptake, localization, and phototoxicity. Increasing numbers of the PEG groups in the mono-, di-, and tri-PEG chlorin conjugates increased the water solubility and sensitivity to hydrolysis and uptake into the ovarian cancer cells. The PEG chlorin conjugates accumulated in the cytoplasm and mitrochondria, but not in lysosomes. Higher phototoxicity was roughly correlated with higher numbers of PEG groups, with the tri-PEG chlorin conjugate showing the best overall ovarian cancer cell photokilling of the series. Singlet oxygen lifetimes, solvent deuteration, and the effects of additives azide ion and d-mannitol were examined to help clarify the photokilling mechanisms. A Type-II (singlet oxygen) photosensitized mechanism is suggested for the di- and tri-PEG chlorin conjugates; however, a more complicated process based in part on a Type-I (radicals or radical ions) mechanism is suggested for the parent chlorin e(6) and the mono-PEG chlorin conjugate.
Project description:Photodynamic therapy (PDT), consisting of photosensitizer, light, and oxygen has been used for the treatment of various diseases including cancers, microbial infections and skin disorders. In this study, we examined the anti-inflammatory effect of chlorin e6-mediated PDT in P. acnes-infected HaCaT cells using photosensitizer chlorin e6 (Ce6) and halogen light. The live and heat-killed P. acnes triggered an upregulation of inflammatory molecules such as iNOS, NO, and inflammatory cytokine in HaCaT cells and mouse model. Ce6-mediated PDT notably downregulated the expression of these inflammatory molecules in vitro and in vivo. Similarly, chlorin e6-mediated PDT was capable of regulating inflammatory response in both live and heat killed S. epidermidis exposed HaCaT cells. Moreover, phosphorylation of p38, JNK, and ERK were reduced by Ce6-mediated PDT. Ce6-mediated PDT also reduced the phosphorylation of IKK?/?, I?B? and NF?B p65 in P. acnes-stimulated HaCaT cells. In addition, the dramatic increase in the nuclear translocation of NF?B p65 observed upon stimulation with P. acnes was markedly impaired by Ce6-based PDT. This is the first suggestion that Ce6-mediated PDT suppresses P. acnes-induced inflammation through modulating NF?B and MAPKs signaling pathways.
Project description:In this work, we analyze the latest data on the molecular docking of a range of SARS-CoV-2 proteins to protoporphyrin IX, verteporfin, and chlorin e6, as well as consider the prospects for using chlorins and porphyrins as agents for photoinactivation of the SARS2 virus.
Project description:Approaches to achieve site-specific and targeted delivery that provide an effective solution to reduce adverse, off target side effects are urgently needed for cancer therapy. Here, we utilized a Trojan-horse-like strategy to carry photosensitizer Chlorin e6 conjugated porous silicon multistage nanovectors with tumor homing mesenchymal stem cells for targeted photodynamic therapy and diagnosis. The inherent versatility of multistage nanovectors permitted the conjugation of photosensitizers to enable precise cell death induction (60%) upon photodynamic therapy, while simultaneously retaining the loading capacity to load various payloads, such as antitumor drugs and diagnostic nanoparticles. Furthermore, the mesenchymal stem cells that internalized the multistage nanovectors conserved their proliferation patterns and in vitro affinity to migrate and infiltrate breast cancer cells. In vivo administration of the mesenchymal stem cells carrying photosensitizer-conjugated multistage nanovectors in mice bearing a primary breast tumor confirmed their tropism toward cancer sites exhibiting similar targeting kinetics to control cells. In addition, this approach yielded in a > 70% decrease in local tumor cell viability after in vivo photodynamic therapy. In summary, these results show the proof-of-concept of how photosensitizer conjugated multistage nanovectors transported by stem cells can target tumors and be used for effective site-specific cancer therapy while potentially minimizing potential negative side effects.
Project description:To improve the tumor-targeting efficacy of photodynamic therapy, biotin was conjugated with chlorin e6 to develop a new tumor-targeting photosensitizer, Ce6-biotin. The Ce6-biotin had good water solubility and low aggregation. The singlet-oxygen generation rate of Ce6-biotin was slightly increased compared to Ce6. Flow cytometry and confocal laser scanning microscopy results confirmed Ce6-biotin had higher binding affinity toward biotin-receptor-positive HeLa human cervical carcinoma cells than its precursor, Ce6. Due to the BR-targeting ability of Ce6-biotin, it exhibited stronger cytotoxicity to HeLa cells upon laser irradiation. The IC50 against HeLa cells of Ce6-biotin and Ce6 were 1.28 µM and 2.31 µM, respectively. Furthermore, both Ce6-biotin and Ce6 showed minimal dark toxicity. The selectively enhanced therapeutic efficacy and low dark toxicity suggest that Ce6-biotin is a promising PS for BR-positive-tumor-targeting photodynamic therapy.