可系统性给药的蜂毒肽脂质纳米颗粒及其抗黑色素瘤作用研究

发布时间：2019-05-28 11:32

【摘要】：蜂毒肽,作为蜂毒的主要成分,是一种具有α螺旋结构的两亲性多肽。蜂毒肽在疾病治疗上有广泛的应用,具有抗炎、镇痛、抗菌、抗病毒、抗辐射和降血压等作用。蜂毒肽应用于肿瘤治疗具有独特优势,能够在质膜上打孔造成细胞裂解死亡,从而避开了肿瘤的耐药性问题。然而,蜂毒肽自身存在的溶血性问题、非特异性作用以及易被代谢降解等缺点,严重阻碍了其在抗肿瘤方面的临床应用。如何在活体内安全有效地运输蜂毒肽抵达肿瘤组织而不产生毒副作用,仍是极具挑战的难题。本文采用以仿HDL多肽脂质纳米颗粒为基础的运载方法,有效地解决了蜂毒肽在活体肿瘤治疗中存在的溶血问题,并延长了其在体内的循环周期,而且实现了在肿瘤区域特异性地释放,主要取得了以下研究结果： (1)发明了一种新型的含蜂毒肽的功能性杂化多肽α-melittin。它是由蜂毒肽与一条α螺旋结构的载脂蛋白模拟肽(α-peptide)通过共价连接而成。α-melittin在保留了蜂毒肽的自发形成多聚体和裂解细胞等属性的基础上,具有更强的磷脂亲和力以及α螺旋结构形成能力。与α-peptide相比,α-melittin具有更强大的粒径控制力,能够将磷脂形成的纳米颗粒粒径控制在10nm左右甚至更小,同时具有很高的包封率(80%)。 (2)利用α-melittin合成的脂质纳米颗粒α-melittin-NP,实现了活体内蜂毒肽的安全有效递送。α-melittin-NPs是一种粒径在14nm左右,分散性良好的球形纳米颗粒,其核心可以同时运载疏水性分子。α-melittin-NPs性质稳定,在血清和血浆等生理溶液中仍然保持稳定的纳米结构。凭借蜂毒肽与α-peptide羧基端疏水性氨基酸的连接设计以及α-melittin与磷脂层的强力结合,α-melittin-NPs有效地隐藏了蜂毒肽的阳离子电荷(21.33±1.64mV),表现为近中性纳米颗粒(2.45±0.56mV)。α-melittin-NPs明显降低了蜂毒肽的溶血性和非特异性破坏作用。体外溶血实验表明,α-melittin-NPs对血细胞的裂解作用显著降低,即使在501μM的高浓度下也只有8%左右的溶血率。细胞增殖实验显示,α-melittin-NPs对细胞生长抑制的IC50值(11.26±1.37μM)较蜂毒肽(1.71±0.04μM)有显著的提升。激光共聚焦连续成像的结果表明,α-melittin-NPs与黑色素瘤细胞接触后造成细胞膜通透性改变,诱导细胞内容物泄漏、细胞核分解,最终导致细胞死亡。Annexin V-FITC/PI的双色标记和流式分析结果表明,α-melittin-NPs导致肿瘤细胞死亡的主要途径是发生坏死,也能诱导少量细胞发生凋亡。最后,α-melittin-NPs通过尾静脉注射至皮下荷瘤鼠体内。经过4次给药治疗,黑色素瘤的生长得到了明显抑制,与对照组相比其抑制率高达82.8%。与此同时,通过对治疗小鼠血液的生理生化指标的检测,以及对脏器的病理切片分析,并未显示α-melittin-NPs对机体产生明显的毒副作用。 (3)研制同步运输紫杉醇和蜂毒肽的超小纳米颗粒(PTX-OL)α-melittin-NPs,具有协同抗黑色素瘤的作用。利用激光共聚焦显微镜观察到α-melittin-NPs在与肿瘤细胞相互作用时,会将蜂毒肽释放至包括质膜在内的膜系统上,而核心内的染料分子则被直接释放至胞浆中。(PTX-OL)α-melittin-NPs在两个治疗机制上充分发挥了紫杉醇和蜂毒肽的抗肿瘤功能。肿瘤细胞增殖实验的结果显示,(PTX-OL)α-melittin-NPs对黑色素瘤杀伤效率明显增强,IC50值是α-melittin-NPs的44.8%。Annexin V-FITC/PI双色标记和流式分析结果表明,相比于α-melittin-NPs,(PTX-OL)α-melittin-NPs能够诱导更多细胞发生坏死,提升幅度为10-20%。而且,在活体黑色素瘤治疗上,(PTX-OL)α-melittin-NPs同样展现出增强的肿瘤生长抑制效果。综上所述,本研究利用仿HDL多肽脂质纳米颗粒作为蜂毒肽的运载体,通过将蜂毒肽隐藏在单层磷脂分子层内,有效地解决了其在体内运输中存在的溶血性和非特异性作用的问题,提高了蜂毒肽在体内的循环半衰期,为在活体内使用蜂毒肽治疗肿瘤提供了有效的工具。采用蜂毒肽与化疗药物协同配合的方法,将会突破单一靶标在肿瘤治疗上的限制,为肿瘤的多靶点治疗带来了新的机遇。
[Abstract]:The bee venom peptide, as the main component of bee venom, is an amphiphilic polypeptide with a polar helix structure. The bee venom peptide has wide application in the treatment of diseases, and has the effects of resisting inflammation, relieving pain, resisting bacteria, resisting virus, resisting radiation and lowering blood pressure, and the like. The bee venom peptide has the unique advantages of being applied to the treatment of the tumor, and the cell lysis and death can be caused by punching on the plasma membrane, so that the drug resistance problem of the tumor is avoided. However, the problems of haemolytic, non-specific and easy to be metabolically degraded by the bee venom peptide have seriously hindered their clinical application in the field of anti-tumor. It is still a challenge to how to safely and effectively transport the bee venom peptide to the tumor tissue without any toxic and side effect. The method has the advantages of effectively solving the hemolysis problem of the bee venom peptide in the treatment of the in-vivo tumor and prolonging the cycle period in the body, and realizing the specific release in the tumor region, The following results were mainly obtained: (1) The invention provides a novel functional hybrid polypeptide-melitti containing a bee venom peptide, N. It is by covalent attachment of a bee venom peptide to an apoprotein mimic peptide of a polar helix. The method has the advantages that, on the basis of the properties such as the spontaneous formation of the multimer and the lysing cell of the bee venom peptide, the antigen-melittin has stronger phospholipid affinity and the formation energy of the dendritic spiral structure, L-melittin has a more powerful particle size control, and the particle size of the nano-particles formed by the phospholipid can be controlled to be about 10 nm or less, and has a very high encapsulation rate (80%, compared to the P-peptide. and (2) using the lipid nano-particles I-melittin-NP synthesized by the chitosan-melittin to realize the safety of the bee venom peptide in the living body, It is a spherical nano-particle with a particle size of about 14 nm and a good dispersivity, and its core can carry water at the same time. Stable, stable in physiological solutions, such as serum and plasma. The cationic charge (21.33-1.64 mV) of the bee venom peptide (21.33-1.64 mV) was effectively hidden by the connection design of the hydrophobic amino acid and the strong binding of the antigen-melittin with the phospholipid layer. MV). The antigen-melittin-NPs significantly reduce the hemolytic and non-specific rupture of the bee venom peptide. The results of in vitro hemolysis showed that the lysis of the blood cells was significantly reduced, even at a high concentration of 501. m u.M, even at a high concentration of 501. m The results of cell proliferation showed that the IC50 value of the inhibition of cell growth (11.26-1.37. mu.M) was significantly higher than that of the melittin (1.71-0.04. mu.M). The results of laser confocal continuous imaging show that the cell membrane permeability changes after contact with melanoma cells, inducing the leakage of the cell contents and the decomposition of the nucleus, leading to the fine formation of the cell membrane. The results of two-color labeling and flow analysis of Annexin V-FITC/ PI showed that the main route of tumor cell death was necrosis, and a small number of cells could be induced by the two-color labeling and flow analysis of Annexin V-FITC/ PI. Apoptosis. Finally, the antigen-melittin-NPs was injected into the subcutaneous tumor by the tail vein. The growth of melanoma was significantly inhibited after 4 administration, and the inhibition rate was as high as 82 in the control group. 8%. At the same time, by the detection of the physiological and biochemical indexes of the blood of the treated mice, and the analysis of the pathological sections of the organs, it is not shown that the antigen-melittin-NPs is toxic to the body. Side effects. (3) Preparation of ultra-small nanoparticles (PTX-OL)1-melittin-NPs for simultaneous transport of paclitaxel and bee venom, with synergistic anti-black The role of the antigen-melittin-NPs in the interaction with the tumor cells is observed by a laser confocal microscope, and the bee venom peptide is released to the membrane system, including the plasma membrane, while the dye molecules in the core are directly released. (PTX-OL)-Melittin-NPs played the role of paclitaxel and melittin in two treatment mechanisms. The results of the experiment of tumor cell proliferation showed that (PTX-OL)-melittin-NPs increased the killing efficiency of melanoma and the IC50 value was 44.8% of the antigen-melittin-NPs. The Annexin V-FITC/ PI two-color marker and the flow analysis showed that, compared with the P-melittin-NPs, (PTX-OL)-melittin-NPs can induce more fine. Necrosis of the cell, the lifting amplitude is 1 0-20%. Also, in the treatment of live melanoma, (PTX-OL)--melittin-NPs also exhibit enhanced tumor growth To sum up, the present study makes use of the HDL-polypeptide lipid nanoparticles as the carrier of the bee venom peptide, and effectively solves the hemolytic and non-special effects of the bee venom peptide in the in-vivo transportation by hiding the bee venom peptide in the single-layer phospholipid molecule layer. The problem of the opposite sex effect improves the circulating half-life of the bee venom peptide in the body, and is a method for treating the tumor by using the bee venom peptide in the living body. The method of the invention can break through the limitation of the single target in the treatment of the tumor, and is a multi-target therapy for the tumor,
【学位授予单位】：华中科技大学
【学位级别】：博士
【学位授予年份】：2015
【分类号】：R943;R96

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