Virus-Like Particles By Electrostatic Assembly Of Triblock Protein Polymer

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.