【作者】 刘磊；

【导师】 王俊有；

【作者基本信息】 华东理工大学 ， 化学工程与技术， 2020， 硕士

【摘要】 病毒是生物界中最简单的生物系统之一,可以特定地向宿主细胞传递遗传物质。由于病毒不确定的传染性和致病性,此类结构作为诊疗载体的应用一直备受限制。模仿天然病毒结构合成的类病毒颗粒不仅可以降低天然病毒递送药物过程中的不确定风险,同时可以构筑多功能纳米载体,在生物医药领域应用广泛。本论文中,我们研究了三嵌段蛋白质聚合物C4-S10-Bk12和聚酰胺-胺(PAMAM)树状大分子电荷组装制备类病毒颗粒。其中,C4是负责提供胶体稳定性的中性氨基酸链段,S100是具有折叠功能的结构嵌段,Bk12是带有12个正电荷的聚赖氨酸组装嵌段。羧基封端的PAMAM树状大分子作为阴离子聚合物。二者基于电荷作用组装制备了结构可控的类病毒颗粒。利用激光光散射(DLS),我们系统探究了组装时间、三嵌段蛋白质聚合物C4-S10-Bk12预组装、PAMAM树状大分子浓度和代数对于组装结构的影响。基于透射电镜(TEM)、原子力显微镜(AFM)和冷冻电镜(Cryo-TEM)表征了类病毒颗粒的三维结构和尺寸大小,最后设计盐滴定实验评估了组装结构的耐盐稳定性。研究发现组装过程首先是从组装嵌段Bk12和PAMAM之间的电荷作用开始,之后是折叠嵌段S10通过β-折叠和疏水堆叠形成颗粒的硬质纤维结构,最后由稳定嵌段C4包裹形成稳定的三层类病毒颗粒,为后续此类颗粒的载体化应用奠定了基础。具体实验结论如下:(1)选用PAMAM G2.5和三嵌段蛋白质聚合物C4-S10-Bk12制备类病毒颗粒G2.5-P,发现组装过程至少需要48小时。48小时内,散射光强增长迅速,之后增长逐渐减缓直至稳定,得到三层结构类病毒颗粒。TEM结果表明,类病毒颗粒长约115 nm(±25 nm),宽约14 nm;由于电荷和折叠嵌段的竞争组装,调控PAMAM树状大分子和三嵌段蛋白质聚合物C4-S10-Bk12的组装比例,降低蛋白质聚合物预组装,可实现类病毒颗粒的高效制备;(2)使用PAMAM G2.5、G3.5、G4.5和G5.5和三嵌段蛋白质聚合物C4-S10-Bk12制备类病毒颗粒,我们通过改变PAMAM分子的代数来控制类病毒颗粒尺寸。AFM结果表明,类病毒颗粒的长度从110 nm(±15 nm)增长到145 nm(±25 nm),宽度从20 nm增长到35 nm,高度平均增长3 nm,PAMAM分子在颗粒内部处于压缩状态。(3)类病毒颗粒G2.5-P、G5.5-P在盐溶液中不稳定,高浓度下由于屏蔽作用,静电驱动力减弱,结构被破坏。更多还原

【Abstract】 As one of the simplest biological systems,viruses are effective vehicles for the delivery of genetic material into susceptible host cells.However,the applications of natural viruses for drug delivery have been limited due to the their indeterminate infectivity and pathogenicity.By mimicking the structure of natural viruses,synthetic virus-like particles(VLPs)can not only reduce the indeterminate risk in the process of delivering drug,but also perform as multi-functional nano-carriers,which are widely used in the field of biomedicine.In this thesis,we investigated the preparation of VLPs by controlled assembly of triblock protein polymer C4-S10-Bk12 and polyamidamide-amine(PAMAM)dendrimers.The C4 block with approximately 100 amino acids provides colloidal stability to the supramolecular structures.The midblock S10 features folding function,and the BK12 block consisting of 12 lysines provides positive charges for the electrostatic assembly with anionic components,which are carboxyl-terminated PAMAM dendrimers in our study.VLPs with controllable structure were prepared based on charge interaction.Dynamic light scattering(DLS)was applied to investigate the assembly process,as well as the effects of assembly time,pre-assembly of triblock protein polymer C4-S10-Bkl2,the concentration and generations of PAMAM dendrimers on assembly.The three-dimensional structure and size of VLPs were characterized by transmission electron microscopy(TEM),atomic force microscopy(AFM)and cryo-electron microscopy(Cryo-TEM).Finally,we tested the stability of the formed particles against increasing salt concentrations.We found that the assembly process began with the charge interaction between the Bk12 block and PAMAM dendrimers.Then the folding block S10 folds cooperatively into beta-rolls that stack into stiff,elongated fibers through hydropHobic stacking.Finally,the C4 block provides colloidal stability to the three-layered VLPs.The experimental results are as follows:(1)VLPs were prepared by the aseembly of PAMAM G2.5 and triblock protein polymer C4-S10-Bk12.It was found that the assembly process needs at least 48 hours.In the first 48 hours,the scattering intensity grow rapidly and reached a plateau after 120 h.Over time,small particles grow into VLPs featuring a clearly three-layers structure with a length around 115 nm(±25 nm),a width around 14 nm;Control the mixing ratio of PAMAM and the triblock protein polymer C4-S10-Bk12 allow to reduced the pre-assembly of the protein polymer,leading to optimal formation of VLPs.This result suggests that the formation of VLPs involves multiple steps and competing processes between the charge assembly and the folding of S10 block.(2)Assembly of PAMAM G2.5,G3.5,G4.5,G5.5 and triblock protein polymer C4-S10-Bk12 forms stable virus-like particles.The particle size including length,width and height increase with increasing PAMAM generation,indicating the comprossed state of the PAMAM dendrimers inside of the VLPs.(3)Upon increasing salt concentration,virus-like particle G2.5-P,G5.5-P dissociate due to strong screening of the electrostatic driving forces,suggesting poor stability of the formed VLPs.更多还原