Polyplex Formed From A Single Network Cationic Polymer Molecule Of Precisely Designed Size And PH-sensitivity

The great therapeutic potential of nucleic acids is their capability to express,silence,or gene editing nearly any targeted genes.However,safe and efficient delivery system is still the need for gene materials to medicines.Virus represents the high efficiency mechanism of gene delivery in nature.Twenty years passed,a series of synthetic gene delivery vectors had been studied,only a small fraction of these have progressed into clinical trials.None of these vectors has received FDA approval,the main bottleneck in resolving the delivery is that ideal non-viral carrier should accomplish five tasks:(A)condensing siRNA into nanoparticles;(B)deliver the nanoparticles to targeted cells;(C)endosomal escape from endosome;(D)releasing siRNA in cytoplasm;and(E)self-metabolize into nontoxic molecules.Based on the knowledge above,this study focuses on a functionally tailorable synthetic carrier formed by series thermodynamically self-consisting assembly and easy to formulate.This system consists of a polyplex core made of one molecule of networked cationic polymer of uniform size and a uni-lamella shell of a rationally designed tri-block copolymer.Nucleic acids can be packed tightly inside the network cavity,and released readily in response to cellular pH by degradation of the aromatically conjugated imine linkages of the polymer backbone.Cell targeting agents may be conjugated to the distal end of shell-forming copolymer and self-assembled in optimized population.This system may serve as a universal vehicle for non-covalently attaching variety of delivery components.Cationic polyimines based on spermine have generated interest as efficient,biodegradable polymeric carriers for gene delivery.These polymers could be stable in physiological conditions but degrade immediately in acidic environment.What' more,polyimines were easy to produce,bio-responsive degradable,facilitate endosome escape,cytoplasm release and self-metabolism.Previously,we reported PSI,the first one of them had been demonstrated for successful deliver siRNA and is nontoxic.However,the relation of polyamines' structures and siRNA intracellular delivery had not well studied.To improve siRNA delivery efficiency,more copies of siRNAs should be translated into the cytoplasm.What' more,the polymers condensed nucleus acids took place with surface interaction and the nucleus acids within the polyplexes were easy to replace by the macromolecules with negative charge,which made the polyplexes very unstable in vitro and in vivo.Size of polyplexes and molecular weight distribution for bio-degradable cationic polymers were other considerations for synthesized polymers in gene delivery.To address these problems,we reported a convenient method to formulate a polymeric siRNA carrier of tailorable uniform size and pH-responsiveness,comprising a self-consistent polymerization process for convergent molecular weight and aqueous phase self-assembly of required functional components.This carrier system consisted of a cationic polymeric core to achieve the intracellular trafficking and a protective shell for intercellular recognition.The 3D structured core-forming cationic polymer was synthesized from branched and linear amino-group bearing building blocks through aromatically conjugated imine linkages in aqueous solution,for which the diffuse double layer formed around the growing polymer functioned as the molecular weight-regulating factor.The obtained cationic polymer packed siRNA into its intra-molecular cavity(adjusted by the length and fraction of the linear building block)to form a highly condensed single polymer-molecule polyplex of defined size.This study is categorized into three parts.The first part investigate the structurally similar polyimines showed unique performance in dynamic balance in response to the changes of pH mimicking within cellular environment and the difference in the capability in condensing siRNA,uptaking by cells and gene silencing,on account of the pKa of aromatic rings and steric conformations of dialdehyde groups position.Secondly,based on the result,PSI is chose as the small linear linker molecules to design a unique cross-link structure of a cationic polymer,polymerizing with branch low molecular weight PEI,which could contain siRNAs inside the caves of polymer and coat siRNAs as a positive charge membrane to remain uniform size when complex with siRNAs.The results indicate that because of the positive charges of the caves,the siRNAs could stay in the caves and the polymers functioned as a cationic membrane outside.So this unique polyplexes will remained uniform molecular state.If the doses of siRNA increased and the caves inside the polymers were full,siRNAs will bind on the surface and decrease the zeta potential of the polymers subsequently,and then the polyplexes stay instable and aggregated.Finally,the physicochemistry characterizations of the single polyplexes were measured and the gene silencing efficiency was investigated.