纳米金在雄性生殖毒理和雄性避孕中的功能研究
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摘要
纳米材料中的纳米金(AuNPs)已经被应用于诊断影像学、药物载体、肿瘤的光热治疗等领域。最近,关于AuNPs作为多种药物载体进行靶向性治疗肿瘤方面的应用备受亲睐。随着AuNPs在生物医学领域中的应用增加,关于AuNPs在生物体内的命运和潜在毒性问题引起了人们的广泛关注。
进年来,已有研究表明AuNPs能诱导肝脏的氧化损伤、急性炎症和细胞凋亡。相比其他器官系统来说,雄性生殖系统是一个对外界物质较为敏感、易受损的系统。然而,关于AuNPs对生殖毒性的研究,例如AuNPs在生殖器官中的分布,是否能通过生殖系统保护性屏障-血睾屏障(BTB),这些方面的问题仍然未知。我们合成了两种不同化合物修饰的纳米金,甲氧基化聚乙二醇修饰的纳米金(mPEG@AuNP)和氨基化聚乙二醇修饰的纳米金(PEG-NH2@AuNP),通过尾静脉注射成年雄性ICR小鼠。采用电感耦合等离子体质谱(ICP-MS)定量证明了纳米金能够在雄性小鼠睾丸中富集,在各检测时间点发现PEG-NH2@AuNP在小鼠睾丸中的富集量是mPEG@AuNP的两倍以上。通过暗场显微镜观察到小鼠的曲细精管的管腔中存在纳米金,说明纳米金可以通过血睾丸屏障。透射电子显微镜(TEM)证明了纳米金能通过血睾屏障并进入生精细胞。通过连续的三次重复尾静脉注射1mPEG@AuNP和PEG-NH2@AuNP到成年雄性ICR小鼠,检测小鼠的体重、激素水平、睾丸的组织形态学和精子发生过程中减数分裂同源染色体之间的联会和重组交换的研究,以及通过纳米金暴露雄性小鼠和正常雌鼠的交配的研究,证明纳米金没有明显的生殖毒性。然而,我们发现连续的三次重复尾静脉注射PEG-NH2@AuNP,能够上调小鼠的睾酮的水平,结果提示PEG-NH2@AuNP可能存在潜在毒性,提示在应用纳米金时,表面修饰是很重要的方面。
研究表明纳米金没有明显的生殖毒性,我们下一步计划研究纳米金是否能在雄性生殖系统有所应用。已有研究报道表明,金纳米棒在特定波长的近红外光(NIR)的照射下能够产生局部高热。有一些研究者应用金纳米棒的光热转换效应做肿瘤的光热治疗,取得了很好的效果。同时,雄性睾丸中的精子细胞是对温度很敏感的细胞,已有报道关于热水浴杀精用于避孕方面的研究。我们结合这两方面的研究工作,旨在利用金纳米棒的光热效应解决流浪动物的避孕问题。首先,我们合成了甲氧基化聚乙二醇修饰的金纳米棒(GNR),该GNR能特异的将808纳米NIR的光能转化为热能。然后,睾丸原位注射GNR,通过控制GNR剂量和NIR的功率来实现对睾丸处的温度变化控制。最后的研究发现,在较低温度下(37℃,40℃),小鼠的睾丸部分破坏,在7天时,小鼠的生育能力下降到约10%；在60天后,小鼠的生育能力恢复到约50%。在较高温度下(45℃),小鼠的睾丸全部破坏,在7天时,小鼠的生育能力下降到约0,以后不能恢复生育力。我们提供了一个创伤小、可控的雄性避孕方法。该方法方便、有效、廉价、易操作等优点于一身,可以被选做宠物和流浪动物的节育,为下一步发展新型男性避孕技术提供了重要的依据。
Nanomaterials such as gold nanoparticles (AuNPs) are already being used in the fields of diagnostic imaging, drug delivery, and cancer photothermal therapy. In recent times, AuNPs have emerged as attractive candidates for delivery of various drug payloads to their targets. The increasing biomedical applications of AuNPs have raised major concerns regarding their in vivo fate and potential toxic effects in living organisms.
Recently, AuNPs have been shown to induce oxidative stress and acute inflammation and cell apoptosis in the liver. The male reproductive system is known to be more sensitive to exogenous materials than other organ systems and has been classified as vulnerable in recent years. However, a great deal of additional information on the reproductive toxicity of gold nanoparticles, such as the distribution of AuNPs in reproductive organs and their ability to pass through reproduction-relevant physiological barriers [e.g. the blood-testis barrier (BTB)], are still unknown. We synthesize two surface-modified AuNPs, that is co-methoxy and ω-aminoethyl poly(ethylene glycol) capped AuNPs (mPEG@AuNP and PEG-NH2@AuNP). we intravenously injected mPEG@AuNP and PEG-NH2@AuNP and control (HEPES buffer, HBS) via the tail vein into adult male ICR mice. The accumulation of AuNPs in testes was analyzed by inductively coupled plasma mass spectrometry (ICP-MS). The results showed that both mPEG@AuNP and PEG-NH2@AuNP can accumulate in the testes. The Au content of PEG-NH2@AuNP in testes was over two times higher than that of mPEG@AuNP at each time point. The dark field microscope images showed gold grains were located in the lumen of the seminiferous tubules, indicating that mPEG@AuNP can penetrate the BTB. By using TEM, we found that mPEG@AuNP was packed in vesicles of spermatogonia. Male ICR mice received three i.v injections of HBS, mPEG@AuNP and PEG-NH2@AuNP via the tail vein given every other day. We investigated the body weight, hormone levels, testes morphology, meiotic homologous chromosome synapsis and recombination. As well as, we mated the normal female mice with gold nanopartcles exposed male mice. The results showed there were no significant reproductive toxicity with gold nanoparticles treatment. However, we found male ICR mice received three i.v injections of PEG-NH2@AuNP, which elevated the testosterone levels of mice. The results indicated that PEG-NH2@AuNP might have a little toxicity. When we use the AuNPs, we should be care the surface modification of AuNPs.
The results above showed AuNPs have no significant reproductive toxicity. The next plan is to investigate the application of AuNPs in male reproductive system. There are many reports showed that gold nanorods can absorp specific wavelength near infrared light (NIR) producing local hyperthermia. The studies used the photothermal effect of gold nanorods to cure tumors, which have obtained gold effect. Meanwhile, the sperm cells in male testes are temperature sensitive cells. There are some studies reported hot bath can kill the sperms, which were used in male contraception. We combined the two aspects studies, to reslove the contraception of stray animals with photothermal effect of gold nanorods. Firstly, we synthesized methoxy poly(ethyleneglycol)-modified gold nanorods (here abbreviated GNR), which can transfer the light energy of specific808nm infrared light (NIR) to hyperthermal energy. Secondly, we showed that in situ testicular injection of GNR in male mice, and we controlled the testes temperature by the dose of GNR and irradiation power of NIR. Finally, In a lower hyperthermia treatment, the morphology of testes and seminiferous tubules is only partly injured, and fertility indices are decreased to～10%at day7, then recovered to～50%at day60. In a higher hyperthermia treatment, the morphology of testes and seminiferous tubules are totally destroyed, and fertility indice sare decreased to0at day7, unrecovered forever. Herein, we have reported a less invasive and controllable procedure formale contraception. This method is more convenient, efficient,and cheaper than surgical castration, which can be chosen to treat the over population of pets and stray animals. The method is a significant basis for developing a new male contraception technology.
进年来,已有研究表明AuNPs能诱导肝脏的氧化损伤、急性炎症和细胞凋亡。相比其他器官系统来说,雄性生殖系统是一个对外界物质较为敏感、易受损的系统。然而,关于AuNPs对生殖毒性的研究,例如AuNPs在生殖器官中的分布,是否能通过生殖系统保护性屏障-血睾屏障(BTB),这些方面的问题仍然未知。我们合成了两种不同化合物修饰的纳米金,甲氧基化聚乙二醇修饰的纳米金(mPEG@AuNP)和氨基化聚乙二醇修饰的纳米金(PEG-NH2@AuNP),通过尾静脉注射成年雄性ICR小鼠。采用电感耦合等离子体质谱(ICP-MS)定量证明了纳米金能够在雄性小鼠睾丸中富集,在各检测时间点发现PEG-NH2@AuNP在小鼠睾丸中的富集量是mPEG@AuNP的两倍以上。通过暗场显微镜观察到小鼠的曲细精管的管腔中存在纳米金,说明纳米金可以通过血睾丸屏障。透射电子显微镜(TEM)证明了纳米金能通过血睾屏障并进入生精细胞。通过连续的三次重复尾静脉注射1mPEG@AuNP和PEG-NH2@AuNP到成年雄性ICR小鼠,检测小鼠的体重、激素水平、睾丸的组织形态学和精子发生过程中减数分裂同源染色体之间的联会和重组交换的研究,以及通过纳米金暴露雄性小鼠和正常雌鼠的交配的研究,证明纳米金没有明显的生殖毒性。然而,我们发现连续的三次重复尾静脉注射PEG-NH2@AuNP,能够上调小鼠的睾酮的水平,结果提示PEG-NH2@AuNP可能存在潜在毒性,提示在应用纳米金时,表面修饰是很重要的方面。
研究表明纳米金没有明显的生殖毒性,我们下一步计划研究纳米金是否能在雄性生殖系统有所应用。已有研究报道表明,金纳米棒在特定波长的近红外光(NIR)的照射下能够产生局部高热。有一些研究者应用金纳米棒的光热转换效应做肿瘤的光热治疗,取得了很好的效果。同时,雄性睾丸中的精子细胞是对温度很敏感的细胞,已有报道关于热水浴杀精用于避孕方面的研究。我们结合这两方面的研究工作,旨在利用金纳米棒的光热效应解决流浪动物的避孕问题。首先,我们合成了甲氧基化聚乙二醇修饰的金纳米棒(GNR),该GNR能特异的将808纳米NIR的光能转化为热能。然后,睾丸原位注射GNR,通过控制GNR剂量和NIR的功率来实现对睾丸处的温度变化控制。最后的研究发现,在较低温度下(37℃,40℃),小鼠的睾丸部分破坏,在7天时,小鼠的生育能力下降到约10%；在60天后,小鼠的生育能力恢复到约50%。在较高温度下(45℃),小鼠的睾丸全部破坏,在7天时,小鼠的生育能力下降到约0,以后不能恢复生育力。我们提供了一个创伤小、可控的雄性避孕方法。该方法方便、有效、廉价、易操作等优点于一身,可以被选做宠物和流浪动物的节育,为下一步发展新型男性避孕技术提供了重要的依据。
Nanomaterials such as gold nanoparticles (AuNPs) are already being used in the fields of diagnostic imaging, drug delivery, and cancer photothermal therapy. In recent times, AuNPs have emerged as attractive candidates for delivery of various drug payloads to their targets. The increasing biomedical applications of AuNPs have raised major concerns regarding their in vivo fate and potential toxic effects in living organisms.
Recently, AuNPs have been shown to induce oxidative stress and acute inflammation and cell apoptosis in the liver. The male reproductive system is known to be more sensitive to exogenous materials than other organ systems and has been classified as vulnerable in recent years. However, a great deal of additional information on the reproductive toxicity of gold nanoparticles, such as the distribution of AuNPs in reproductive organs and their ability to pass through reproduction-relevant physiological barriers [e.g. the blood-testis barrier (BTB)], are still unknown. We synthesize two surface-modified AuNPs, that is co-methoxy and ω-aminoethyl poly(ethylene glycol) capped AuNPs (mPEG@AuNP and PEG-NH2@AuNP). we intravenously injected mPEG@AuNP and PEG-NH2@AuNP and control (HEPES buffer, HBS) via the tail vein into adult male ICR mice. The accumulation of AuNPs in testes was analyzed by inductively coupled plasma mass spectrometry (ICP-MS). The results showed that both mPEG@AuNP and PEG-NH2@AuNP can accumulate in the testes. The Au content of PEG-NH2@AuNP in testes was over two times higher than that of mPEG@AuNP at each time point. The dark field microscope images showed gold grains were located in the lumen of the seminiferous tubules, indicating that mPEG@AuNP can penetrate the BTB. By using TEM, we found that mPEG@AuNP was packed in vesicles of spermatogonia. Male ICR mice received three i.v injections of HBS, mPEG@AuNP and PEG-NH2@AuNP via the tail vein given every other day. We investigated the body weight, hormone levels, testes morphology, meiotic homologous chromosome synapsis and recombination. As well as, we mated the normal female mice with gold nanopartcles exposed male mice. The results showed there were no significant reproductive toxicity with gold nanoparticles treatment. However, we found male ICR mice received three i.v injections of PEG-NH2@AuNP, which elevated the testosterone levels of mice. The results indicated that PEG-NH2@AuNP might have a little toxicity. When we use the AuNPs, we should be care the surface modification of AuNPs.
The results above showed AuNPs have no significant reproductive toxicity. The next plan is to investigate the application of AuNPs in male reproductive system. There are many reports showed that gold nanorods can absorp specific wavelength near infrared light (NIR) producing local hyperthermia. The studies used the photothermal effect of gold nanorods to cure tumors, which have obtained gold effect. Meanwhile, the sperm cells in male testes are temperature sensitive cells. There are some studies reported hot bath can kill the sperms, which were used in male contraception. We combined the two aspects studies, to reslove the contraception of stray animals with photothermal effect of gold nanorods. Firstly, we synthesized methoxy poly(ethyleneglycol)-modified gold nanorods (here abbreviated GNR), which can transfer the light energy of specific808nm infrared light (NIR) to hyperthermal energy. Secondly, we showed that in situ testicular injection of GNR in male mice, and we controlled the testes temperature by the dose of GNR and irradiation power of NIR. Finally, In a lower hyperthermia treatment, the morphology of testes and seminiferous tubules is only partly injured, and fertility indices are decreased to～10%at day7, then recovered to～50%at day60. In a higher hyperthermia treatment, the morphology of testes and seminiferous tubules are totally destroyed, and fertility indice sare decreased to0at day7, unrecovered forever. Herein, we have reported a less invasive and controllable procedure formale contraception. This method is more convenient, efficient,and cheaper than surgical castration, which can be chosen to treat the over population of pets and stray animals. The method is a significant basis for developing a new male contraception technology.
引文
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