兰州大学机构库 >生命科学学院
脂肪酸修饰的两亲性多肽自组装机制以及载药能力的研究
Alternative TitleStudy on the Self-Assembly Mechanism of Fatty Acid-Modified Amphiphilic Peptides and Drug Delivery Ability
郭晨
Subtype硕士
Thesis Advisor彭雅丽
2020-05-27
Degree Grantor兰州大学
Place of Conferral兰州
Degree Name理学硕士
Degree Discipline生物化学与分子生物学
Keyword肺癌 肿瘤多药耐药 P-糖蛋白 干扰RNA 药物运输系统 多肽自组装 纳米胶束
Abstract在我国恶性肿瘤中肺癌是发病率和死亡率最高的一种癌症。化疗是肺癌的主要治疗手段。然而和其他癌症一样,在长期服药后产生的肿瘤多药耐药严重阻碍了肺癌的治疗。利用siRNA抑制癌细胞中过表达的P糖蛋白(P-gp)是解决肿瘤多药耐药有效途径之一。但siRNA在应用上面临着一项重要挑战,就是如何才能将siRNA安全有效地递送至肿瘤组织以及高效的细胞转运。此外化疗药物本身具有难溶性、易降解、毒副作用等缺点限制了其临床应用,解决这些问题的有效途径之一也在于开发新型的药物运输系统(DDS)。在诸多药物载体中多肽聚合物胶束因其特殊的壳-核结构,能够同时满足以上两种需求,且具备优良的生物相容性,因此成为我们研究的目标。 在本文中我们以NPS为母肽进行脂肪酸修饰,设计合成了六种两亲性多肽类似物,并命名为M1~6。研究发现其中M2、M3、M4、M6在良好的生物安全性的前提下,不仅具有很强的细胞膜穿透能力,而且能够在水溶液中以极低浓度快速发生自组装形成亲水外壳-疏水内核的纳米胶束,因此具备了药物载体的研究潜力。我们通过圆二色谱检测出M2、M3、M4、M6的二级结构皆为&alpha-螺旋结构,根据对应关系我们判定&alpha-螺旋结构是这四种多肽类似物自组装能力的关键因素之一。同时我们利用动态光散射技术(DLS)发现M2、M3、M4、M6形成纳米胶束的粒径范围主要分布在100~200nm之间。而Zeta电势的检测结果则表明纳米胶束的结构稳定,且外侧带有大量正电荷。我们通过TEM成像观察纳米胶束的具体形态时,发现M2和M6形成的都是纳米颗粒结构,而M3和M4形成的则都是纳米纤维结构。根据自组装强度、入胞能力以及细胞毒性等指标,我们分别选择了综合性能较好的M4和M6进行了体外P-gp siRNA的结合和运载实验。 根据琼脂糖电泳结果显示M4和M6都能够有效结合siRNA,其中又以M4的结合曲线显示出更好的结合能力。又考虑到细胞质膜的安全性,我们最终选择了M4作为P-gp siRNA的载体,在人腺癌肺泡基底上皮细胞A549中进行了运载实验。共聚焦和细胞流式的结果显示M4能够高效的携带荧光素标记的siRNA进入细胞,且比阳性对照脂质体Lipo2000表现出更优质的运载siRNA的潜力。同时实时定量PCR实验结果也表明M4携带P-gp siRNA进入A549细胞后,能够在24h和48h内有效减少了A549细胞中P-gp 相关mRNA的含量,抑制了P-gp的表达。不过其抑制效果与Lipo2000相比却没有显示出对应的优势,我们推测这可能是两个原因导致的,一是M4携带P-gp siRNA进入细胞后的内体逃逸效率弱于Lipo2000,导致siRNA大量被溶酶体降解二是可能在于M4与siRNA之间的结合作用过强影响了siRNA在胞质中的释放,阻碍了其发挥作用。根据以上推测,一来我们利用多种内吞作用抑制剂研究确认了M4的入胞机制,为以后在进行内体逃逸改善实验时提供依据二来我们也根据肿瘤细胞中特殊的氧化还原条件,将M4进一步改造为M4-S-S-C18。与M4相比,M4-S-S-C18主肽链通过两个二硫键相连的半胱氨酸(Cys)与硬脂酸C18相连。期望在肿瘤细胞内存在的大量还原型谷胱甘肽能够快速断裂二硫键,破坏胶束完整性,从而达到加快siRNA释放的目的。此外,聚合物胶束具有特殊的壳-核结构,能够作为一种有效运输难溶性抗癌药物的载体,因此我们在运载siRNA实验的同时也尝试了M4和M4-S-S-C18对阿霉素的包载实验。
Other AbstractLung cancer (LC) is the most common type of cancer in China. Chemotherapy is the main treatment method for lung cancer. But just like other cancers, multi-drug resistance(MDR) of tumors is an important factor limiting LC treatment after long-term medication. The use of siRNA to inhibit over-expressed P-glycoprotein (P-gp) in cancer cells is one of the effective ways to solve MDR. However, one key challenge for the applications of siRNA is the safe and effective delivery of siRNA to the tumor and sufficient cytosolic transport. In addition, the shortcomings of chemotherapeutic agents, such as poor solubility, easy degradation, and toxicity, limit chemotherapeutics application. One of the effective ways to solve these problems is to develop new drug delivery systems (DDS). Polymer micelles which have a special "shell-core"structure, is a perfect carrier to transport hydrophobic drugs and siNRA, so we chose it for our research. In this paper, six amphiphilic peptide analogs named M1~6 were synthesized with NPS as the parent peptide for fatty acid modification. The study showed that under the premise of good biological safety, M2, M3, M4 and M6 not only have strong cell membrane penetration ability but also rapidly self-assemble to form nanomicelles of "hydrophilic outer shell and hydrophobic inner corein aqueous solution at very low concentration. Thus we think they can be used as drug carriers. The secondary structures of M2, M3, M4, and M6 were determined by circular dichroism measurements to be the &alpha-helix. According to the corresponding relationship, we think that the structure of &alpha-helix is one of the key factors for the self-assembly ability of amphiphilic peptides. The characterization of nano-micelles is determined by dynamic light scattering technology (DLS) and the results showed size distribution of particles formed by M2, M3, M4, and M6 is between 100 and 200 nm. And the results of Zeta-potential measurement indicate that the structure nanomicelles is very stable and there are a large amount of positive charges in the hydrophilic shell. We observed the morphology of nanomicelles by TEM, and found that the nanomicelles formed by M2 and M6 are nanoparticle, while M3 and M4 are nanofiber. Finally, on the basis of self-assembly ability, the infiltration efficiency and cytotoxicity, M4 and M6 are selected for the binding and delivery of P-gp siRNA in vitro. According to the results of agarose electrophoresis, both M4 and M6 could bind siRNA, and the M4 showed better binding ability. Considering the safety of cell plasma membrane, we finally chose M4 as the carrier of P-gp siRNA, and performed the delivery experiment in A549 cells. The results of confocal and cell flow cytometry showed that M4 can carry siRNA into cells effectively, and showed a higher delivery efficient than Lipo2000. Meanwhile, the results of real-time quantitative PCR experiments showed that after M4 carried P-gp siRNA into A549 cells, the content of P-gp related mRNA was obvious reduced in A549 cells within 24h and 48h, which means the expression of P-gp was inhibited. However, its inhibitory effection did not show corresponding advantages compared with Lipo2000, we speculated that there are two reasons. First, the endosomal escape efficiency of M4 is weaker than Lipo2000, which causes a large amount of siRNA is degraded by lysosomes. The second reason is that the excessive binding between M4 and siRNA affects the release of siRNA in the cytoplasm and and prevents its function. Based on our speculations, firstly, we used a variety of endocytosis inhibitors to confirm the entry mechanism of M4, and provide basis for further studySecondly, according to the special REDOX conditions in tumor cells, M4 was further modified into M4-S-S-C18. Compared with M4, the main peptide chain of M4-S-S-C18 is connected to stearic acid C18 through disulfide bonds of two cysteine (Cys). It is expected that the large amount of reduced glutathione present in tumor cells can quickly break disulfide bonds and destroy the integrity of micelles, so as to accelerate the release of siRNA. In addition, the polymer micelles have a special "shell-core"structure, which can be used as an effective carrier for the transport of insoluble anticancer drugs. Therefore, while performing siRNA delivery experiments we also tried encapsulation experiments of Adriamycin by M4 and M4-S-S-C18.
Pages69
URL查看原文
Language中文
Document Type学位论文
Identifierhttps://ir.lzu.edu.cn/handle/262010/466755
Collection生命科学学院
Affiliation
生命科学学院
First Author AffilicationSchool of Life Sciences
Recommended Citation
GB/T 7714
郭晨. 脂肪酸修饰的两亲性多肽自组装机制以及载药能力的研究[D]. 兰州. 兰州大学,2020.
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