PH-responsive DNA Nano-hydrogel For MRNA Delivery And Expression

The delivery of mRNA to manipulate protein expression has become a current hot spot and has attracted widespread attention,becoming an important emerging approach for vaccine preparation and drug development,since that mRNA overcomes the problem of infection and mutation risks in transgenes and can work as drugs for the treatment of diseases.Although there are currently some vehicles that deliver mRNA into cells,they have not yet reached a good balance in terms of expression efficiency and biocompatibility.Here,we develop a DNA nano-hydrogel system for mRNA delivery.The nano-hydrogel is all composed of DNA except the target mRNA,so it has superior biocompatibility compared with those chemical vehicles.In parallel,the nano-hydrogel can be compacted into a nanosphere under the crosslinking by well-designed "X"-shaped DNA scaffolds and DNA linkers,facilitating the delivery into cells through endocytosis.In addition,smart intracellular release of the mRNA is achieved by incorporating a pH-responsive i-motif structure into the nano-hydrogel.Thus,taking the efficient delivery and release together,mRNA can be translated into the corresponding protein with a high efficiency,which is comparable to that of the commercial liposome but with a much better biocompatibility.The methodology has been validated by successful delivering mRNA encoding Gaussia luciferase(a secreted protein)and enhanced green fluorescent protein(an intracellular protein),respectively.Due to the excellent biocompatibility and efficiency,this nano-hydrogel system is expected to become a competitive alternative for delivering functional mRNA either in vitro and in vivo.The work of this paper mainly includes the following parts:1.Construction of pH-responsive nano-hydrogelWhether the DNA nano-hydrogel system can be successfully constructed will affect its function,so we verified its assembly.The system is based on DNA,loading mRNA,so that mRNA can be delivered into the cell and express the encoded protein.The system is designed and adopts DNA linkers with i-motif to cross-link the target mRNA and the "X"-shaped DNA scaffolds to form a nano-hydrogel with a compact nanosphere structure.The assembly of the "X"-shaped DNA scaffolds and DNA linkers was verified by electrophoresis,and the results showed that the "X"-shaped DNA scaffolds and DNA linkers can be assembled.Next,the pH response ability of "X"-shaped DNA scaffolds and DNA linkers was explored,and the results showed that under acidic conditions,"X"-shaped DNA scaffolds and DNA linkers disintegrated,and under neutral or alkaline conditions,"X"-shaped DNA scaffolds and DNA linkers can be assembled smoothly.2.Intracellular delivery of pH-responsive nano-hydrogel loaded mRNAIn order to evaluate the ability and biological safety of nano-hydrogel to delivery mRNA,we first explored the distribution of mRNA in the cell based on the intracellular laser confocal microscope imaging system.Then,flow cytometry was used to analyze the absorption capacity of the cells to the nano-hydrogel to further confirm the ability of the nano-hydrogel to carry mRNA.Then,we explored the cellular uptake mechanism of nano-hydrogel,and the results show that nano-hydrogel is mainly through clathrin-mediated endocytosis and a small amount of nano-hydrogel is through lipid raft-caveolae endocytosis into the cell.Next,the mechanism of mRNA lysosomal escape was explored,and the results showed that in the presence of the inhibitors,the mechanistic target of rapamycin complex 1(m TORC1)signaling pathway was inhibited,and the release of mRNA was suppressed correspondingly.The results consisted with some previous reports,which showed that the transporter located in the endocytic compartment allows mRNA to flow out across the lysosome and participates in the activation of the m TORC1 signaling pathway.Finally,we studied the toxic effects of nano-hydrogel on cells.The flow cytometry apoptosis experiment was used to compare the effects of liposomes and nano-hydrogel on the apoptosis of He La cells.The results showed that as the amount of delivered mRNA increased,the apoptosis rate of the mRNA delivery group using liposomes also increased,while the apoptosis rate of the group using nanohydrogels hardly changed.The same conclusion can be drawn from the results of CCK-8.Overall,these results highlight the safety advantages of using nano-hydrogel to deliver mRNA,and indicate that nano-hydrogel are expected to be used in areas where large amounts of mRNA need to be delivered,laying a foundation for subsequent expression of mRNA carried in cells.3.Intracellular expression of pH-responsive nano-hydrogel loaded mRNAThen we further explored the expression of mRNA carried by the nano-hydrogel in the cell.Enzyme activity determination,RT-PCR,Western blot and other analytical methods show that the pH-responsive nano-hydrogel can successfully deliver the loaded mRNA into the cell and express the corresponding protein at a high level.The expression level is comparable to that of commercial lipids.In terms of delivery and expression effects,a comparative study with pH-unresponsive nano-hydrogel shows that the pH-responsive nano-hydrogel delivery system we constructed has a good ability to release mRNA,so that it can express synthetic protein.In addition,studies have also shown that the nano-hydrogel delivery system has considerable universality,applicable to both cancer cells(He La cells)and normal cells(human liver cells L02),and is suitable for different types of loaded mRNA(Gaussia luciferase mRNA and enhanced green fluorescent protein mRNA)can also be expressed,which is a promising mRNA delivery system.Therefore,the DNA nano-hydrogel system we constructed has the ability to deliver the carried mRNA into the cytoplasm and express the encoded protein,and the delivery system has good stability and versatility.