免疫调控药物纳米金刚石-CpG寡脱氧核苷酸复合物的研究

发布时间：2018-02-02 12:47

本文关键词： 纳米金刚石 CpG ODN 免疫刺激活性抗肿瘤效应　出处：《中国科学院研究生院(上海应用物理研究所)》2016年博士论文　论文类型：学位论文

【摘要】：治疗型核酸,包括功能化修饰的寡脱氧核苷酸(ODNs),适配体(aptamers),小干扰RNA(siRNA),在一系列疾病(从遗传性到后天获得的疾病以及癌症)的治疗中显示出很强的应用前景。未甲基化的胞嘧啶-磷酸盐-鸟嘌呤寡脱氧核苷酸(CpG ODNs)就是其中一种具有免疫刺激活性的核酸,最初发现于病毒及细菌的DNA中。人工合成的CpG ODNs是一种很好的免疫佐剂,在抗感染、癌症以及过敏性疾病的中有广泛的应用。CpG ODNs的临床实验目前处于Ⅰ-Ⅲ期。然而,一些副作用的显现使其无法继续研究。这些副作用主要与高剂量的重复给药相关,因为单链CpG的细胞摄取率很低且很容易被代谢清除。因此,急需发展一种新的输运系统不但可以提高细胞的摄取率而且可以抵抗核酸酶的降解发挥长时间的免疫刺激效应。纳米材料作为控释的药物输运载体有广阔的应用前景。各种各样的纳米材料,从无机、有机到生物大分子都被用作CpG的载体刺激细胞产生相应的免疫反应。然而,高昂的费用及潜在的毒性效应限制了其在体内的应用。纳米材料的一系列载体中,纳米金刚石(NDs)拥有卓越的载运能力、灵活的表面可修饰性以及良好的生物相容性。特别是NDs在生理条件下可以自发形成多孔团簇结构,对于载运小分子化疗药物及生物大分子显示出“海绵”效应。这篇工作,我们采用多聚赖氨酸(PDL)修饰的功能化的纳米金刚石(fNDs)用于细胞内及体内CpG寡核苷酸的输运并且探讨其免疫刺激活性及潜在的抗肿瘤效应。主要研究内容和结果如下:(1)首先设计CpG ODN载体,用阳离子聚合物PDL修饰原始的NDs,形成fNDs复合物;随后通过静电相互作用吸附带负电的CpG,形成CpG-fNDs复合物。原始NDs的平均粒径为247nm,zeta电位为33.0eV;当包裹了带正电荷的PDL之后,fNDs的平均粒径增加到325nm,zeta电位增加到67.3eV;fNDs静电吸附了CpG之后,平均粒径进一步增大,达到338nm;然而Zeta电位则降低为40.6eV。(2)然后,我们使用激光共聚焦荧光显微镜评估cpg-fnds的细胞摄取。摄取时间为6h,细胞内平均荧光强度定量分析显示:与单链cpg相比,cpg-fnds组的细胞摄取率提高了将近3个数量级;之后研究摄取的cpg-fnds在细胞内的清除,结果显示在最初的6h摄取孵育结束后,24,48,72h细胞内的平均荧光强度分别是单链cpgodn组的900倍,830倍以及400倍。(3)接下来,我们研究了cpg-fnds的体外免疫刺激活性。结果发现cpg-fnds孵育raw264.7细胞6h,可以显著的促进肿瘤坏死因子(tnf-α)以及白介素-6(il-6)的释放。与单链cpg组相比,tnf-α和il-6的释放量分别提高到59倍及20倍。之后检测了长时间(24h,48h,72h)免疫刺激因子的释放,发现cpg-fnds组72h仍然可以检测到tnf-α和il-6的释放,释放量分别为单链cpg组的13倍及26倍。体外实验数据表明,cpg-fnds有长期的免疫刺激活性,在细胞水平可以持续3天。(4)为了进一步评估cpg-fnds潜在的治疗效应,我们又将免疫刺激研究进行到体内。icr小鼠尾静脉注射cpg-fnds之后测定小鼠血清中释放的免疫因子的表达量变化。结果发现在尾静脉注射3h后可以显著地诱发血清中il-12和il-6的释放。与单链cpg组相比,il-12和il-6的释放量分别提高了19倍以及61倍。接下来,我们研究了其长期的免疫刺激效果,发现48h的释放量仍有单链cpg组的4倍。体内实验同样证明,cpg-fnds有长期的免疫刺激活性,在动物水平可以持续2天。(5)既然免疫刺激效果如此好,那么在体内是否安全呢?我们使用近红外荧光染料xenolightcf750共价标记nds,监测其在体内生物分布和清除。结果发现,分布在各器官的nds在72小时会彻底清除。主要脏器病理切片及肝功能无明显变化,这些结果证实cpg-fnds在体内非常安全。(6)最后,我们评估了cpg-fnds的在tlr9阳性的肿瘤模型(b16-f0和4t1)的抗肿瘤效应。结果发现cpg-fnds可以显著的抑制肿瘤的生长,对于b16-f0和4t1肿瘤模型,抑瘤率分别为73.3%和28.4%。总之,我们开发了一种新颖的治疗型核酸输送系统,可以提高cpgodn的摄取,在体内外均有缓释效应,而且cpg-fnds复合物在小鼠肿瘤模型的成功应用,为癌症的免疫治疗提供了新的思路,更有可能将其设计并应用于纳米医药领域。
[Abstract]:Treatment of type of nucleic acid, including oligonucleotide functionalized (ODNs), aptamer (aptamers), small interfering RNA (siRNA), in a series of diseases (to get acquired from hereditary disease and cancer) showed a strong application prospects. In the treatment of unmethylated cytosine phosphate guanine oligodeoxynucleotide (CpG ODNs) is one kind of immunostimulatory nucleic acid, originally found in viruses and bacteria in DNA. CpG synthetic ODNs is a good adjuvant in anti infection, clinical trials have been widely used in.CpG ODNs cancer and allergic diseases in the present in the I - III period. However, some side effects appear so that it can not continue to study. These side effects mainly with high doses of repeated administration, because the cellular uptake of single stranded CpG rate is very low and it is easy to be metabolic clearance. Therefore, it is urgent to develop an The new transport system can not only improve the degradation of cell uptake and resistance to nuclease play a long time. The immune stimulatory effect of nano materials as drug controlled release carrier transport have broad application prospects. All kinds of nano materials, from inorganic to organic and biological macromolecules have been used as the carrier of CpG stimulation the cell has the corresponding immune response. However, the high cost and potential toxicity limits its in vivo application. A series of carrier nano materials, nano diamond (NDs) has excellent carrying capacity, flexible surface modification and good biocompatibility. Especially in physiological NDs under the condition of the spontaneous formation of porous cluster structure for carrying small molecule agents and biological macromolecules show "sponge" effect. This work, we use poly lysine (PDL) functionalized The nano diamond (fNDs) for cells and in vivo CpG oligonucleotide transport and to investigate its immune activity and antitumor effect of potential stimulation. The main research contents and results are as follows: (1) the design of CpG ODN vector, the original NDs modified with cationic polymer PDL, the formation of the fNDs complex; followed by electrostatic interaction. The adsorption of charged CpG, the formation of the CpG-fNDs complex. The original NDs average particle size was 247nm, the zeta potential is 33.0eV; when the package with the positive charge of PDL, the average particle size of the fNDs increased to 325nm, the zeta potential increased to 67.3eV; after fNDs electrostatic adsorption of CpG, the average particle size increase, reach 338nm; however, the Zeta potential is reduced to 40.6eV. (2) and then, cellular uptake we use laser confocal fluorescence microscope to evaluate the cpg-fnds. The uptake time is 6h, the mean fluorescence intensity of cells in quantitative analysis showed that with single stranded CPG Compared with the cellular uptake rate in cpg-fnds group was increased by nearly 3 orders of magnitude; after study the uptake of cpg-fnds in cells was clear, results show that at the end of the 6h uptake after the initial incubation, the average fluorescence intensity of 24,48,72h cells was 900 times of single stranded cpgodn group, 830 times and 400 times (3). Next, we studied the in vitro immune stimulating activity of cpg-fnds. The results showed that cpg-fnds of RAW264.7 cells incubated with 6h could significantly promote tumor necrosis factor alpha (tnf- alpha) and interleukin -6 (IL-6) release. Compared with the single CpG group, the release amount of tnf- alpha and IL-6 were increased to 59 times and 20 times. After the detection of long time (24h, 48h, 72h) immune stimulating factor release, cpg-fnds group 72h still can be detected by tnf- alpha and IL-6 release, the release amount of single stranded CpG group respectively 13 times and 26 times in vitro. The experimental data show that cpg-fnds has the long-term immunity thorn Activated, can last for 3 days at the cellular level. (4) in order to further evaluate the therapeutic effect of cpg-fnds potential, we will study the immune stimulation in.Icr mice after intravenous injection of cpg-fnds for determination of expression of release of immune factors in serum of mice. The results found in the tail vein after injection of 3H significantly IL-12 the IL-6 induced serum release. Compared with the single stranded CpG group. The release amount of IL-12 and IL-6 were increased 19 times and 61 times. Next, we investigated the long-term immune stimulating effect, found that the release amount of 48h is 4 times of single stranded CpG group. In vivo experiments also showed that cpg-fnds has long immunostimulatory activity, in animal level can be continued for 2 days. (5) since the immune stimulating effect is so good, so in the body is safe? We use the near infrared fluorescent dye xenolightcf750 NDS monitoring in the covalent labeling. Biodistribution and clearance. The results showed that the distribution in various organs of the NDS will be completely cleared in 72 hours. The main pathological sections and liver function had no obvious change, these results suggest that cpg-fnds is safe in vivo. (6) finally, we evaluated cpg-fnds in TLR9 positive tumor models (B16-F0 and 4T1). Anti tumor effect. The results showed that cpg-fnds can significantly inhibit tumor growth for B16-F0 and 4T1 tumor model, the inhibition rate was 73.3% and 28.4%. respectively. In conclusion, we developed a novel therapeutic nucleic acid delivery system, can improve the uptake of cpgodn, both in vitro and in vivo release effect, and the cpg-fnds complex in success application of mouse tumor model, provides a new way for cancer immunotherapy, are more likely to be designed and used in nano medicine.

【学位授予单位】：中国科学院研究生院(上海应用物理研究所)
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R943;TB383.1

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