GnRH介导MLT纳米微粒靶向治疗前列腺癌

The current treatments for the advanced prostate cancer are still far from satisfactory, especially when progress to castrate-resistant prostate cancers (CRPC), they fail to respond to androgen-deprivation therapy (ADT). Both the domestic and foreign researches have shown that the melittin (MLT), derived from honeybee venom protein, has cytotoxicity to prostate cancer cells. This project explores a novel target therapy for prostate cancer with melittin (MLT) nano-particles derived from honeybee venom protein. MLT nano-particles are prepared from MLT proteins encapsulated by the phospholipids nanometer carrier and subsequently coated with gonadotropin releasing hormone (GnRH) by chemical conjugation method. Because GnRH receptors are highly expressed in both androgen-dependent and castrate-resistant prostate cancers, the MLT nano-particles can specifically target all prostate cancers regardless of androgen status. After binding to GnRH receptors on the cell membrane, the MLT tetramers penetrate the cell membrane and cause cancer cells disintegration and death. Moreover, the application of nanotechnology makes it much easier for the MLT particles to transverse the blood barriers and dramatically increases the efficacy of the treatment. The MLT nano-particles can also specifically target the cells containing GnRH receptors in the pituitary and testis, therefore, results in a permanent medical castration. Our study may identify MLT nano-particles as a novel targeted therapy for prostate cancers, particularly the advanced and castrate-resistant prostate cancers.

前列腺癌的治疗现状仍不令人满意,尤其是当癌细胞转化成去势抵抗性时,雄激素全阻断治疗的疗效甚微。国内外的实验研究已表明蜂毒蛋白（MLT）对前列腺癌细胞有杀伤、抑制和细胞毒作用。本课题拟将MLT经磷脂纳米载体包裹制成MLT纳米微粒，然后用化学耦联的方法将促性腺激素释放激素（GnRH）结合到该纳米微粒表面，使其具有GnRH介导的高度靶向性。因前列腺癌细胞无论雄激素依赖性或去势抵抗性，均高表达GnRH受体，GnRH介导的MLT纳米微粒能特异性地和前列腺癌细胞结合，通过蜂毒蛋白四聚体洞穿细胞膜而造成癌细胞的崩解。此外，纳米技术的应用使得该纳米微粒更易通过血脑屏障和血睾屏障，破坏脑垂体及睾丸中高表达GnRH受体的细胞，提高其与GnRH受体的结合率，形成永久性的药物去势，有望为前列腺癌，特别是晚期前列腺癌、去势抵抗性前列腺癌探索出一种新的治疗方法。

本项目以二硬脂酰磷脂酰胆碱(DSPC)、胆固醇（70:30）、二硬脂酰磷脂酰乙醇胺－聚乙二醇2000吡啶二硫代丙酰胺（DSPE-PEG(2000)PDP）、二硬脂酰磷脂酰乙醇胺－聚乙二醇2000胺（DSPE-PEG(2000)Amine）为膜材，采用薄膜分散法成功制备蜂毒素长循环脂质体。合成的蜂毒素长循环脂质体直径约170nm。通过溶血实验证实，蜂毒长循环脂质体溶血性较蜂毒素显著降低，达到了课题设计的目的。MTT实验，平板克隆实验，Transwell实验的结果表明，蜂毒素长循环脂质体对PC3前列腺癌细胞有显著的的细胞毒性，增殖抑制以及侵袭抑制作用，而且效果与单独应用蜂毒素相当。流式细胞仪检测以及TUNEL实验结果表明，蜂毒素长循环脂质体能显著增加PC3前列腺癌细胞的凋亡。蛋白印迹试验结果也表明，经蜂毒素长循环脂质体处理后的PC3前列腺癌细胞内凋亡相关蛋白表达显著增加。Akt和mTOR蛋白可定位于细胞线粒体膜，其下游通道蛋白是调节细胞生物学活动重要的信号通路。对比未经处理的PC3细胞和经蜂毒素长循环脂质体及单独蜂毒素处理的PC3细胞信号通路蛋白的表达水平发现，蜂毒素长循环脂质体能显著抑制PI3k/Akt/mTOR通路相关蛋白的磷酸化，进而抑制PC3前列腺癌细胞增殖，促进其凋亡，而且效果与单独应用蜂毒素相当。上述结果表明，合成的蜂毒素长循环脂质体最大程度上保留了蜂毒素的抗肿瘤活性。随后的动物实验进一步证明了蜂毒素长循环脂质体可靠的体内抑瘤活性。ELLISA法对不同组别裸鼠重要脏器蜂毒素浓度进行检测，可以发现，相较于直接注射蜂毒素，应用蜂毒素长循环脂质体能显著增加裸鼠体内移植瘤的蜂毒素浓度，并减缓其代谢速度，使肿瘤局部持续处于较高浓度的蜂毒素环境中，以达到更好的抑瘤效果。这很好地证实了蜂毒素长循环脂质体良好的靶向性。本项目结果表明，GnRH修饰的蜂毒素长循环脂质体在促进药物蓄积、抑制肿瘤生长等方面优于游离蜂毒素制剂，而且能够有效降低蜂毒素的溶血性。因此，本研究为设计用于肿瘤治疗的纳米药物递送系统提供了一些启示。