五氮齿稀土配合物对小鼠腹水肝癌细胞及其实体瘤的光动力效应

Photodynamic Effects of Pentaazadentate Macrocyclic Rare earth Complexes on AH Hepatoma in Mice

目的 研究五氮齿稀土配合物（ＰＲＥＣ）光敏剂对昆明小鼠腹水肝癌ＡＨ 细胞株在体外及体内的光动力效应，为评价其作为一类第二代新型光动力治疗试剂提供实验依据。方法 选用五氮齿羧钆（ＰＧｄＣＯＯＨ）、五氮齿钆（ＰＧｄ）、五氮齿铕（ＰＥｕ）和五氮齿钐（ＰＳｍ） 四种自行合成的ＰＲＥＣ光敏剂，用ＭＴＴ法测定它们对ＡＨ细胞的暗毒性和在不同浓度、以波长７６０ ｎｍ 红光（ 功率密度１４ ｍＷ／ｃｍ２） 在不同照光时间条件下，对体外培养的小鼠腹水肝癌ＡＨ细胞的光动力效应；用流式细胞技术测定了ＰＧｄ 光敏作用对ＡＨ 细胞群体时相分布的影响；观察了荷ＡＨ实体瘤（ 平均体积为２２０ ｍｍ３）小鼠尾静脉注射ＰＧｄＣＯＯＨ、ＰＥｕ 或ＰＳｍ（ 剂量均为１２ ｍｇ／ｋｇ） 后２４ ｈ，用７３０～８００ ｎｍ 红光（ 功率密度为１０ ｍＷ／ｃｍ２） 照光，每次１ ｈ，每天１ 次，连续３ 天后肿瘤体积的变化，比较肿瘤长到照光前１０ 倍体积时所需时间的差别，并计算每组小鼠的平均生存寿命。另外还观察了ＰＧｄＣＯＯＨ、ＰＥｕ 和ＰＳｍ 对健康小鼠皮肤的光毒性作用。结果 ＰＲＥＣ的浓度低于１－０ μｍｏｌ／Ｌ时，ＡＨ细胞存活率高达９３％ 以上；

Objective To investigate the photodynamic effects of four pentaazadentate rare earth complexes (PREC) on AH hepatoma cells in vitro and in vivo and to evaluate their value as a potential novel second generation photosensitizer in photodynamic therapy (PDT) of cancers.Methods Four PREC (PSm, PEu, PGd and PGdCOOH) were synthesized in our laboratory, which showed an intense absorption (lg ε>4 ) at 766 772 nm in the spectral window of PDT. The surviving rates of AH ascites hepatoma cell in vitro were determined by MTT method after photodynamic treatment with red light of 730 800 nm irradiation (14 mW·cm -2 ) for different irradiation time and in the absence or presence of PREC in various concentration. Dark toxicities of PREC in different concentration for AH ascites hepatoma cells in vitro were also examined. The transplanted AH solid tumors in the hind left leg of Kunming mice were treated by PDT 7 10 days after inoculation, in which four PRECwere injected respectively i.v. in a dose of 12 mg·kg -1 of body weight prior to light exposure and the irradiation was carried out at 10 mW·cm -2 for 1 h per day for consecutive 3 days. The tumor volumes were measured using a hemielliposoidal model daily in the first 4 days and then 3 times per week. The tumor volumes and regrowth delay times were compared with those of the control group. The skin phototoxicities of PREC for healthy mice in vivo with the same irradiation times and sensitizer doses as used in the AH solid tumors groups were also examined using a 5 scale criterion, in which normal skin response was rated on a scale from no effect (0) to extensive edema with moist exudate and loss of toes (5). Results The surviving fractions of AH ascites hepatoma cells in vitro were calculated after photodynamic treatment, in which the 50% of AH cells killing needs about 30 min of irradiation for 6 μmol/L of PGd in DMSO and the irradiation times were shorted to 23 and 15 min for 10 and 30 μmol/L of PGd, respectively. During the 15 min of irradiation the photodynamic effects in vitro were obvious by using 30 μmol/L of PREC and 14 mW·cm -2 of red light irradiation. The sequence of efficacy was PGdCOOH>PGd>PEu>PSm. The surviving fractions of AH cells for PREC in the concentration of 1 0 to 30 0 μmol/L in dark were variable from 93% to 85%. The surviving fraction varied for AH cells at different phases of cell cycle before and after 30 min of irradiation in the presence of PGd, in which the ratio of G 2/G 1 reached to 4 95, indicating that the photodamage ability to AH cells appeared different at different phases of cell cycle. The tumor necrosis, tumor shrinkage and a delay of the tumor regrowth were observed after PDT treatment. Photodynamic effect on the tumors were obvious. Tumor regrowth was delayed by about 4-7 days depending on different sensitizers used and the average lives of the experimental mice were about 6-7 weeks, which were almost 10 days longer than that of control mice. The sequence of PDT of PREC for solid tumors was PGdCOOH>PEu>PSm(P<0 01 ). The phototoxicities of PREC for the healthy mice in vivo were on the scale between 1-2, which indicated slight edama with erythema. Conclusions Our findings indicate that the pentaazadentate rare earth complexes showing strong killing effects on AH carcinoma cells in vitro and in vivo may be considered as a promising candidate of second generation photosensitizer for photodynamic therapy of tumors and worthy further investigation.