Shiho Hirohara

In Vitro Heavy-Atom Effect of Palladium(II) and Platinum(II) Complexes of Pyrrolidine-Fused Chlorin in Photodynamic Therapy

Introduction of a heavy atom into photosensitizers generally facilitates intersystem crossing and improves the quantum yield (Phi(Delta)) of singlet oxygen ((1)O(2)), which is a key species in photodynamic therapy (PDT). However, little information is available about the physiological importance of this heavy-atom effect. The aim of this study is to examine the heavy-atom effect in simple metallochlorins in vitro at the cellular level. 1,3-Dipolar cycloaddition of azomethine ylide to 5,10,15,20-tetrakis(pentafluorophenyl)porphyrinato palladium(II) and platinum(II) afforded metallochlorins 4b and 4c in yields of 17.1 and 12.9%, respectively. The Phi(Delta) values increased in the order of 4a (0.28) < 4b (0.89) < 4c (0.92) in C(6)D(6). The photocytotoxicity of 4a, 4b, and 4c was evaluated in HeLa cells at a light dose of 16 J x cm(-2) with lambda > 500 nm and increased in the order of 4a < 4b < 4c at the concentration of 0.5 microM. The photocytotoxicity of 4b and 4c was significantly inhibited by addition of sodium azide, but not D-mannitol, suggesting that (1)O(2) is the major species causing cell death. Our results clearly indicate that 4b and 4c act as efficient (1)O(2) generators due to the heavy-atom effect in a cellular microenvironment as well as in nonphysiological media.

Synthesis and Photocytotoxicity of S-Glucosylated 5,10,15,20-Tetrakis(tetrafluorophenyl)porphyrin Metal Complexes as Efficient 1O2-Generating Glycoconjugates

5,10,15,20-Tetrakis(4-(2,3,4,6-tetra-O-acetyl-beta-d-glucopyranosylthio)-2,3,5,6-tetrafluorophenyl)porphyrin 2a and its Zn(II), Pd(II), and Pt(II) complexes 2b, 2c, and 2d were prepared in excellent yields by nucleophilic substitution of the corresponding free-base porphyrin and metalloporphyrins with acetyl 2,3,4,6-tetra-O-acetyl-1-thio-beta-d-glucopyranoside. Deprotection of 2a, 2b, 2c, and 2d by alkaline hydrolysis afforded the corresponding S-glucosylated porphyrin 3a and its metal complexes 3b, 3c, and 3d. The structures and purity of all new photosensitizers were confirmed by elemental analysis and (1)H, (13)C, and (19)F NMR, UV-vis, and steady-state luminescence spectroscopy. The relative efficiency of singlet oxygen ((1)O(2)) production increased in the order of free-base fluoroporphyrins (2a and 3a) < Zn(II) complexes (2b and 3b) < Pd(II) complexes (2c and 3c), which can be explained in terms of the heavy-atom effect, while the (1)O(2)-producing efficiency of Pt(II) complexes (2d and 3d) were exceptionally low. In vitro photocytotoxicity of these eight S-glucosylated photosensitizers was examined in HeLa cells. Although all protected photosensitizers 2a, 2b, 2c, and 2d showed no photocytotoxicity, the photosensitizers 3a, 3b, and 3c exerted potent photocytotoxicity. These results clearly indicated that the sugar moieties of 3a, 3b, and 3c act as not only water-solubility-enhancing functionalities but also cellular-uptake-enhancing elements. Photocytotoxicity testing of 3a, 3b, and 3c in the presence of reactive oxygen species inhibitors suggested that (1)O(2) is the major mediator of cell death. Hence, the Zn(II) and Pd(II) complexes 3b and 3c are promising photosensitizers having cellular uptake-facilitating units (sugar moieties) and enhanced (1)O(2) generation due to the heavy-atom effect.

Cellular Uptake and Photocytotoxicity of Glycoconjugated Porphyrins in Hela Cells.

Thirty‐two glycoconjugated porphyrins were synthesized by a modification of Lindsey method in the presence of Zn(OAc)2.2H2O as a template. The Zn2+ ion template strategy improved the yield about three‐fold in the case of meta ‐substituted tetraphenylporphyrins. In addition, free‐base porphyrins were obtained almost quantitatively by demetalation with 4 M HCI. Sixteen deacetylated glycoconjugated porphyrins were tested as candidate photodynamic therapy (PDT) drugs using HeLa cells. Most of the deacetylated glycoconjugated porphyrins showed higher cellular uptake than tetraphenylporphyrin tetrasulfonic acid (TPPS), and 5,10,15, 20‐tetrakis[4‐(β‐D‐arabinopyranosyloxy)phenyl]porphyrin p‐5d) in particular showed 18.5‐fold higher uptake than TPPS. The photocytotoxicity of 5,10,15,20‐tetrakis[4‐(β‐D‐glucopyranosyloxy) phenyl]porphyrin (p‐5a), (p‐5d and TPPS was examined with HeLa cells, using a light dose of 16 J/cm2. These photosensitizers had no cytotoxicity in the dark, but their photocytotoxicity increased in the order of TPPS p‐5a p‐5d. These results suggest p‐5d is a good candidate for a PDT drug.

Hydrophobicity parameters (Log P) of glycoconjugated porphyrins for photodynamic therapy evaluated by reversed phase HPLC

Photodynamic therapy (PDT) is one of the most effective treatments for cancer. We synthesized and characterized a series of 16 glycoconjugated porphyrins using a modification of the Lindsey method in the presence of Zn(OAc)2·2H2O as a template. The Zn2+ ion template strategy improved the yield about 3-fold in the case of meta-substituted tetraphenylporphyrins, and free-base porphyrins were obtaind almost quantitatively by demetalation with 4 M HCl. The hydrophobicity parameter (Log P) of these porphyrins was evaluated by reversed phase high-performance liquid chromatography (RP-HPLC). The Log P values ranged from +4.8 to +7.8, so that most of the compounds are amphiphilic. The effect of the glycopyranosyl unit on the hydrophobicity of these compounds is discussed on the basis of the Log P value.

Aqueous RAFT synthesis of block and statistical copolymers of 2-(α-D-mannopyranosyloxy)ethyl methacrylate with 2-(N,N-dimethylamino)ethyl methacrylate and their application for nonviral gene delivery

Block copolymers composed of 2-(α-D-mannopyranosyloxy)ethyl methacrylate (ManEMA) and 2-(N,N-dimethylamino)ethyl methacrylate (DMAEMA) were synthesized by aqueous RAFT polymerization. The number-average degree of polymerization (DPn) of ManEMA segments was constant at 33, and the DPn of DMAEMA segments varied from 98 to 241. Statistical copolymers with a similar composition were also prepared via aqueous RAFT polymerization. The mannose-presenting nature was evaluated by a turbidimetric assay using Concanavalin A. The clustering rate of statistical copolymers was faster than those of the corresponding block copolymers. By contrast, no significant differences between block and statistical copolymers were found in their DNA-condensing ability as evaluated using gel shift assays and in their cytotoxicity in the transfection of plasmid DNA (pEGFP-N1) to HeLa cells. However, the overall transfection efficiency significantly depended on the monomer distribution. Statistical copolymers showed an overall transfection efficiency comparable to those of poly(DMAEMA·HCl)s, but block copolymers showed no detectable transfection under the same conditions.

Synthesis, Photophysical Properties, and Biological Evaluation of trans-Bisthioglycosylated Tetrakis(fluorophenyl)chlorin for Photodynamic Therapy.

trans-Bisthioglycosylated tetrakis(fluorophenyl)chlorin (7) was designed as a powerful photodynamic therapy (PDT) photosensitizer based on the findings of our systematic studies. We show here that the trans-bisthioglycosylated structure of 7 enhanced its uptake by HeLa cells and that the chlorin ring of 7 increased the efficiency of reactive oxygen species generation under the standard condition of our photocytotoxicity test. The versatility of 7 in PDT treatment was established using weakly metastatic B16F1 melanoma cells, metastatic 4T1 breast cancer cells, the RGK-1 gastric carcinoma mucosal cell line, and three human glioblastoma cell lines (U87, U251, and T98G). The pharmacokinetics of 7 in mice bearing 4T1 breast cancer cells showed a high tumor-to-skin concentration ratio (approximately 60) at 24 h after intraperitoneal injection. The PDT efficacy of 7 in vivo was approximately 250-times higher than that of mono-l-aspartyl chlorin e6 (9) in mice bearing 4T1 breast cancer cells.

Encapsulation of rat bone marrow stromal cells using a poly-ion complex gel of chitosan and succinylated poly(Pro-Hyp-Gly).

Encapsulation of stem cells into a three‐dimensional (3D) scaffold is necessary to achieve tissue regeneration. Prefabricated 3D scaffolds, such as fibres or porous sponges, have limitations regarding homogeneous cell distribution. Hydrogels that can encapsulate cells such as animal‐derived collagen gels need adjustment of the pH and/or temperature upon cell mixing. In this report, we fabricated a poly‐ion complex (PIC) hydrogel of chitosan and succinylated poly(Pro–Hyp–Gly) and assessed its effect on cell viability after encapsulation of rat bone marrow stromal cells. PIC hydrogels were obtained successfully with a concentration of each precursor as low as 3.0–3.8 mg/ml. The maximum gelation and swelling ratios were achieved with an equal molar ratio (1:1) of anionic and cationic groups. Using chitosan acetate as a cationic precursor produced a PIC hydrogel with both a significantly greater gelation ratio and a better swelling ratio than chitosan chloride. Ammonium succinylated poly(Pro–Hyp–Gly) as an anionic precursor gave similar gelation and swelling ratios to those of sodium succinylated poly(Pro–Hyp–Gly). Cell encapsulation was also achieved successfully by mixing rat bone marrow stromal cells with the PIC hydrogel simultaneously during its formation. The PIC hydrogel was maintained in the culture medium for 7 days at 37°C and the encapsulated cells survived and proliferated in it. Although it is necessary to improve its functionality, this PIC hydrogel has the potential to act as a 3D scaffold for cell encapsulation and tissue regeneration. Copyright

Syntheses, photophysical properties, and photocytotoxicities of tetrakis(fluorophenyl)porphyrin derivatives bearing 2-hydroxyethylthio groups.

Porphyrin derivatives for photodynamic therapy are frequently modified with hydrophilic groups to improve their water solubility; however, such hydrophilic groups not only improve the solubility but also affect the photodynamic behavior of the compound. The suitable number and pattern of the hydrophilic substituents depend on the nature of the hydrophilic groups. In this article, we explore the optimum architecture for 5,10,15,20-tetrakis(pentafluorophenyl)porphyrin (TFPP) derivatives bearing 2-hydroxyethylthio substituents. All five derivatives, namely mono-, cis-bis-, trans-bis-, tris- and tetrakis-substituted TFPP, were successfully synthesized by the nucleophilic aromatic substitution of TFPP with 2-hydroxyethanethiol, separated, and subsequently identified using ESI-TOF mass spectrometry and (1)H and (19)F NMR spectroscopies. The hydrophilicity of the compounds increased with an increase in the number of 2-hydroxyethylthio groups. The singlet oxygen and hydroxyl radical generation efficiencies were estimated using chemical probes following photoirradiation (λ>500nm). trans-Bissubstituted TFPP exhibited the highest efficiency for both singlet oxygen and hydroxyl radical generation. The photocytotoxicities of the photosensitizers were evaluated in HeLa cells following photoirradiation (λ>500nm, 16Jcm(-2)), and increased with an increase in number of 2-hydroxyethylthio groups. In the case of 2-hydroxyethylthio-substituted TFPPs, the fully substituted TFPP was the most efficient architecture plausibly because of the result of the hydrophilicity of the compound rather than a greater efficiency in the generation of reactive oxygen species.

Synthesis, photophysical properties, and photodynamic activity of positional isomers of TFPP-glucose conjugates

The synthesis and characterization of a complete set of positional isomers of tetrakis(perfluorophenyl)porphyrins (TFPP)-glucose conjugates (1OH, 2OH, 3OH, 4OH, and 6OH) are reported herein. The cellular uptake and photocytotoxicity of these conjugates were examined in order to investigate the influence of location of the TFPP moiety on the d-glucose molecule on the biological activity of the conjugates. An In vitro biological evaluation revealed that the certain of these isomers have a greater effect on cellular uptake and cytotoxicity than others. The TFPP-glucose conjugates 1OH, 3OH, and 4OH were found to exert exceptional photocytotoxicity in several types of cancer cells compared to 2OH and 6OH substituted isomers.