Intracellular Delivery And Function Of Small Non-Coding RNAs

Small non-coding RNAs are RNAs that are no longer than 40 nt in length and are not involved in protein coding.Although these RNA molecules are small,they play a vital role in the regulation of almost all life processes including cell proliferation,differentiation,apoptosis,cell metabolism,and immunity.Some small non-coding RNAs,such as microRNAs(miRNAs)and small interfering RNAs(siRNAs),which can regulate intracellular target genes,are considered to have potential application value in clinical treatment of diseases such as cancer.This paper focuses on these two types of small non-coding RNAs,and develops new methods for intracellular delivery of miRNAs or siRNAs and real-time monitoring of their intracellular functions.Due to the negative electricity of miRNA mimics,miRNA inhibitors and siRNA,they can not directly enter cells through cell membranes to perform their functions,and are easily degraded by nucleases widely existing in organisms.Therefore,the development of non-coding RNA delivery vehicles to achieve efficient delivery of non-coding RNA into cells while protecting its degradation is of great significance for the development of non-coding RNA-based disease therapy.On the other hand,while delivering small non-coding RNAs into target cells,simultaneous monitoring the function of these small non-coding RNAs by using probes can provide important guidance for further optimization of the delivery vector.To this end,this paper mainly focuses on developing new methods for the delivery of miRNA mimics and siRNAs into cancer cells and probes for delivery and function monitoring simultaneously,including:1.Development of cationic cell-penetrating peptides for targeted delivery ofmiRNABased on the characteristics of cationic peptides that can self-assemble to form nanocomplexes by electrostatic interaction with miRNAs or siRNAs,we structurally modified the cationic penetrating peptide nona-arginine(R9).Two kinds of targeting groups,RGD cyclic peptide and folic acid were modified to the ends of R9.These two cationic cell-penetrating peptides can self-assemble with miRNAs through electrostatic interaction to form nanocomplexes.We have demonstrated that these two cationic cell-penetrating peptides can not only efficiently deliver miRNAs to the corresponding target cells,but also selectively deliver miRNAs to the target cells in multicellular environment.Therefore,this strategy provides a feasible way to achieve cell selective delivery of miRNAs in a multicellular environment.2.Development of a novel peptide probe for miRNA delivery and functionmonitoringBased on the previous work,we further developed a novel strategy for miRNA delivery and functional monitoring.Based on two kinds of biocompatible reactions,such as copper-catalyzed click reaction and UV-induced photo-click reaction,we developed a new method for constructing multifunctional peptide probes by modifying R9 by orthogonal functional groups.By modifying folic acid and a turn-on fluorescent probes for caspase-3 imaging to both ends of cationic cell-penetrating peptide nona-arginine(R9),we successfully constructed the miRNA delivery and functional monitoring peptide probe,which can can self-assemble with miRNAs through electrostatic interaction to form nanocomplexes.Self-assembled nanocomplexes can efficiently deliver miRNA to specific target cells and realize real-time imaging of the functions of miRNA.3.Development of a carbon dots based reductive responsive miRNA deliverysystemBased on the surface modification of carbon dots,we constructed a miRNA delivery-release system that responds to reducing microenvironments.MiRNAs were modified to the surface of carbon dots by disulfide bonds,which could be reduced in the reducing microenvironment of the cancer cells,thereby achieving controlled release of the miRNA in the cancer cell.This strategy is a complement to the self-assembled nanocomposite transport system.4.Development of an antibody-based UV light-regulated siRNA delivery systemUsing a siRNA that inhibits the expression of programmed death ligand 1(PD-L1),we designed a light-controlled release antibody-siRNA conjugate for delivery of PD-L1 siRNA into tumor cells with high expression of PD-L1.The siRNA of PD-L1 and the PD-L1 antibody Atezolizumab can be covalently linked by a UV irradiation responsible ligand.After the antibody-siRNA conjugate was endocytosed into target tumor cells highly express PD-L1,siRNA can be released from the antibody-siRNA conjugate with UV irradiation.Through this system,the membrane PD-L1 and the endogenous PD-L1 can be blocked simultaneously,which can improve the deficiency of current immunologic checkpoint blocking therapy and improve the effect of immunotherapy.