Polyphenol Promoted Low Molecular Weight Polymers-Mediated RNAi

RNA therapeutics have great potential to treat a wide variety of human diseases including cancer,hereditary diseases and cardiovascular diseases.In contrast to plasmid DNA,siRNA,limited by the rigidity of its double-stranded structure,smaller size and low charge density,is more difficult to condense.As ones of the most widely adopted non-viral vectors for siRNA delivery,various cationic polymers have been developed,however the efficiency-toxicity correlation is unsatisfactory.Polymers with a higher molecular weight produce superior transfection efficacy,but give rise to higher cytotoxicity in the meanwhile,the lower molecular weight counterparts show poor transfection efficacy due to weak interactions with nucleic acids,although they exhibit better cell compatibility.In order to strengthen the complex formation cooperativity and enhance binding ability,low molecular weight polycations were assembled into nanosized supramolecular polymers to enhance binding ability.An alternative strategy is to pre-fabricate siRNAs into nanostructures before complexation with cationic polymers.However,these approaches are not yet satisfactory for clinical translation of siRNA-based therapeutics due to the safety concerns induced by sophisticated siRNA chemical modification and material synthesis.Here,we proposed a novel strategy to fabricate core-shell structured nanoparticles with robust siRNA delivery efficiency.We pre-fabricated the natural polyphenol epigallocatechin gallate(EGCG)with the siRNA to form negative charged nanoparticles,which were further coated with low molecular weight cationic polymers.This supramolecular strategy makes it easier for polymers to condense the siRNA into uniform and stable nanoparticles,resulting in strikingly high gene knockdown efficacy and excellent biocompatibility,providing an avenue to break down the efficiencytoxicity correlation of cationic polymers.Main findings are summarized as follows:(1)Pre-fabrication siRNA with EGCG to yield core nanoparticles facilitates the condensation of siRNA to uniform nanoparticles(GNPs)with low molecular weight polymers in extremely low dose.Compared to the cationic polymers alone,the nanoparticles specifically down-regulate target genes in vitro and in vivo,and efficiently attenuate inflammation in an intestinal injury model.Considering EGCG as a natural green plant extract and the non-toxic low molecular weight,the nanoparticles exhibit excellent biocompatibility.Notably,this strategy is applicable for polymers with different chemical compositions and topologies,which provides a promising polymermediated siRNA delivery platform.(2)Considering the chemical similarity of different RNAi drugs,we introduced this method for the delivery of single-stranded nucleic acids.The interaction of EGCG with single-stranded nucleic acids and the core-shell structure of GNPs improve the stability of single-stranded nucleic acids and achieve highly efficient gene silencing.The supramolecular strategy successfully delivers different types of single-stranded nucleic acids including antisense nucleotides ASO,DNAzymes,microRNAs,regulates the targeted gene expression and modulates cellular biofunctions.(3)Systemic delivery of RNAi drugs is hampered by the barriers such as instability in blood fluids,poor targeted tissue accumulation and possible immunogenicity.Fluorination modification improves stability of GNPs under serum conditions,reduces the leakage of siRNA,and increases the efficiency of gene delivery compared to GNPs.In in vivo experiments,fluorinated GNPs showed elevated liver distribution and did not induce undesired hematological toxicity.These results lay the foundation for the subsequent systemic siRNA delivery for RNAi therapy.Furthermore,this method not only solves the problem of the unstability of GNPs in systemic siRNA delivery,but also enriches the fluorine effect of fluoropolymers in gene delivery.(4)Polyphenol-functionalized low molecular cationic polymers were synthesized for gene delivery,which simplified the formulation of GNPs in gene transfection.The interaction of polyphenol with nucleic acids facilitates condensation of siRNA into uniform nanoparticles and improves the stability of complex.Among these grafted polymers,the catechol-modified polymer exhibited enhanced cellular uptake,facilitated endosomal escape and showed superior gene transfection efficiency.In vivo,catechol-modified polymer effectively silenced target gene,regulated the downstream gene and protein expression,and ultimately attenuated intestinal damage.In summary,polyphenols facilitate nucleic acids condensation with low molecular weight cationic polymers,and the yielding nanoparticles show robust gene silencing efficiency both in vitro and in vivo.The proposed supramolecular strategy breaks the efficiency-toxicity dilemma of cationic polymers,and permits the development of a general and robust platform for gene delivery.