Berberine (Ber) is a herbal alkaloid with pharmacological activity in general and a high anticancer potency in particular. However, due to its low bioavailability, the difficulty in reaching a target and choosing the right dose, there is a need to improve approaches of Ber use in anticancer therapy. In this study, Ber, noncovalently bound to a carbon nanostructure C60 fullerene (C60) at various molar ratios of the components, was explored against Lewis lung carcinoma (LLC). C60–Ber noncovalent nanocomplexes were synthesized in 1:2, 1:1 and 2:1 molar ratios. Ber release from the nanocomplexes was studied after prolonged incubation at different pH with the liquid chromatography–mass spectrometry analysis of free Ber content. Biological effects of the free and C60-complaxated Ber were studied in vitro towards LLC cells with phase-contrast and fluorescence microscopy, flow cytometry, MTT reduction, caspase activity and wound closure assays. The treatment with C60–Ber nanocomplex was evaluated in vivo with the LLC-tumored C57Bl mice. The mice body weight, tumor size, tumor weight and tumor weight index were assessed for four groups, treated with saline, 15 mg C60/kg, 7.5 mg Ber/kg or 2:1 C60-Ber nanocomplex (15 mg C60/kg, 7.5 mg Ber/kg). Ber release from C60–Ber nanocomplexes was promoted with medium acidification. LLC cells treatment with C60–Ber nanocomplexes was followed by enhanced Ber intracellular uptake as compared to free Ber. The cytotoxicity of the studied agents followed the order: free Ber < 1:2 < 1:1 < 2:1 C60–Ber nanocomplex. The potency of cytotoxic effect of 2:1 C60–Ber nanocomplex was confirmed by 21.3-fold decrease of IC50 value (0.8 ± 0.3 µM) compared to IC50 for free Ber (17 ± 2 µM). C60–Ber nanocomplexes induced caspase 3/7 activation and suppressed the migration activity of LLC cells. The therapeutic potency of 2:1 C60–Ber nanocomplex was confirmed in a mouse model of LLC. The tumor growth in the group treated with 2:1 C60–Ber nanocomplex is suppressed by approximately 50% at the end of experiment, while in the tumor-bearing group treated with free Ber no therapeutic effect was detected. This study indicates that complexation of natural alkaloid Ber with C60 may be a novel therapeutic strategy against lung carcinoma.

中文翻译：

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天然生物碱小檗碱与 C60 富勒烯复合物在体外和体内对 Lewis 肺癌的抗肿瘤作用

小檗碱 (Ber) 是一种草本生物碱，具有一般的药理活性，特别是具有很高的抗癌效力。然而，由于其生物利用度低、难以达到目标和选择合适的剂量，需要改进 Ber 在抗癌治疗中的使用方法。在这项研究中，探索了以不同摩尔比的组分与碳纳米结构 C60 富勒烯 (C60) 非共价结合的 Ber 对抗路易斯肺癌 (LLC)。C60-Ber 非共价纳米复合物以 1:2、1:1 和 2:1 的摩尔比合成。用液相色谱-质谱分析游离 Ber 含量，研究了在不同 pH 下长时间孵育后从纳米复合物中释放的 Ber。使用相差和荧光显微镜、流式细胞术、MTT 还原、半胱天冬酶活性和伤口闭合测定法研究了游离和 C60 复合 Ber 对 LLC 细胞的体外生物学效应。在 LLC 肿瘤 C57Bl 小鼠体内评估了 C60-Ber 纳米复合物的治疗。评估四组小鼠的体重、肿瘤大小、肿瘤重量和肿瘤重量指数，用盐水、15 mg C60/kg、7.5 mg Ber/kg 或 2:1 C60-Ber 纳米复合物（15 mg C60/kg， 7.5 毫克 Ber/kg)。中等酸化促进了 C60-Ber 纳米复合物的 Ber 释放。与游离 Ber 相比，用 C60-Ber 纳米复合物处理 LLC 细胞后，Ber 细胞内摄取增强。所研究药物的细胞毒性顺序如下：游离 Ber < 1:2 < 1:1 < 2:1 C60–Ber 纳米复合物。2:1 C60–Ber 纳米复合物的细胞毒性作用效力通过 IC50 值 (0.8 ± 0.3 µM) 比游离 Ber (17 ± 2 µM) 的 IC50 降低 21.3 倍来证实。C60–Ber 纳米复合物诱导 caspase 3/7 活化并抑制 LLC 细胞的迁移活性。2:1 C60–Ber 纳米复合物的治疗效力在 LLC 的小鼠模型中得到证实。在实验结束时，用 2:1 C60–Ber 纳米复合物治疗的组的肿瘤生长被抑制了约 50%，而在用游离 Ber 治疗的荷瘤组中，未检测到治疗效果。该研究表明，天然生物碱 Ber 与 C60 的络合可能是一种新的肺癌治疗策略。C60–Ber 纳米复合物诱导 caspase 3/7 活化并抑制 LLC 细胞的迁移活性。2:1 C60–Ber 纳米复合物的治疗效力在 LLC 的小鼠模型中得到证实。在实验结束时，用 2:1 C60–Ber 纳米复合物治疗的组的肿瘤生长被抑制了约 50%，而在用游离 Ber 治疗的荷瘤组中，未检测到治疗效果。该研究表明，天然生物碱 Ber 与 C60 的络合可能是一种新的肺癌治疗策略。C60–Ber 纳米复合物诱导 caspase 3/7 活化并抑制 LLC 细胞的迁移活性。2:1 C60–Ber 纳米复合物的治疗效力在 LLC 的小鼠模型中得到证实。在实验结束时，用 2:1 C60–Ber 纳米复合物治疗的组的肿瘤生长被抑制了约 50%，而在用游离 Ber 治疗的荷瘤组中，未检测到治疗效果。该研究表明，天然生物碱 Ber 与 C60 的络合可能是一种新的肺癌治疗策略。1 C60–Ber 纳米复合物在实验结束时被抑制了约 50%，而在用游离 Ber 治疗的荷瘤组中未检测到治疗效果。该研究表明，天然生物碱 Ber 与 C60 的络合可能是一种新的肺癌治疗策略。1 C60–Ber 纳米复合物在实验结束时被抑制了约 50%，而在用游离 Ber 治疗的荷瘤组中未检测到治疗效果。该研究表明，天然生物碱 Ber 与 C60 的络合可能是一种新的肺癌治疗策略。