The Self-assembled Nucleic Acid Nanogel As Novel Vectors For Functional Nucleic Acid Delivery

Gene therapy using functional nucleic acids as the drugs is a promising strategy for disease treatment.However,the lack of ideal vectors that can safely and effectively deliver the functional nucleic acid drugs into the target cells is a crucial factor that prevents the translation of gene therapy from fundamental study to clinic use.Particularly,the traditional non-viral vectors usually introduce the cationic groups to interact with negatively-charged functional nucleic acids,subsequently compress the nucleic acids to form delivery complex.Although cationic carriers can effectively compress and protect the functional nucleic acids,they usually exhibt serious biotoxicity,leading to the failure of clinical trials.As the endogenous macromolecular materials,the nucleic acids not only have inherent biocompatibility and biodegradability but also possess unique Watson-Crick base pairing which provides the basis for nucleic acid drug loading.Based on the well-established nucleic acid nanotechnology,herein we designed a series of non-cationic nucleic acid nanogels that contains different functional nucleic acids for in vitro and in vivo delivery.These novel delivery systems avoid the use of conventional cationic carriers,which compress the nucleic acids through electrostatic interaction.Based on nuclec acid self-assembly,we demonstrated that functional nucleic acids can be compressed and protected inside the nanogel,subsequently serving as effective delivery system for target gene regulation.The main contents and results are summarized below:1.A nucleic acid nanogel for effective siRNA delivery and antitumor therapyInspired by cationic carriers that usually compress and protect nucleic acid drugs inside the delivering complexes,we constructed a noncationic nucleic acid nanogel system using siRNAs as crosslinkers,in which the siRNAs can be fully embedded inside to avoid the enzymatic degradation.First,the DNA-grafted polycaprolactone(DNA-g-PCL),synthesized by conjugating dibenzocyclooctyl-modified DNA strands onto the azide-modified polycaprolactone by copper-free click reaction,possessed multivalent crosslinking sites to hybridize with siRNA linkers.Upon simply mixing the siRNA linkers and DNA-g-PCL brushes with different ratios,nanogels can be rapidly assembled with tunable size.Based on the strategy of nucleic acid hybridization,the siRNAs were completely embedded in nanogel,resulting in the enhanced tolerance to nuclease.We further evaluated the cellular uptake and gene silencing of nucleic acid nanogel in vitro,and it was demonstrated that the nucleic acid nanogel could be rapidly internalized by cells,and the gene silencing aroused by siRNA-embedded nanogel was almost comparable with that of Lipofectamine 2000 in vitro.Further,we evaluated the antitumor effect and pharmacokinetic behavior of siRNA-crosslinked nanogel in tumor-bearing nude mice,and the results revealed that the nucleic acid nanogel had longer blood half-life than Lipofectamine 2000 owing to the negative charge of nucleic acid nanogel,resulting in excellent passive targeting ability and antitumor efficacy.2.A nucleic acid nanogel for effective co-delivery of Cas9/sg RNA and gene editingAs an adaptive prokaryotic immune system against exogenous nucleic acids,the CRISPR/Cas9 system that is composed of two macromolecules:Cas9 and single guide RNA(sg RNA),has been rapidly developed into RNA-mediated gene editing platform.One of the major obstacles for its clinical transformation is lack of safe and effective delivery vector to efficiently deliver the giant CRISPR/Cas9 complex into the target cells.So far,the most common carriers of Cas9/sg RNA were cationic vectors which loaded the macromolecular cargos via electrostatic interaction.However,this construction strategy cannot avoid the toxicity of cationic carriers.Based on the strategy of nucleic acid hybridization assembly,a non-cationic nucleic acid delivery system capable of simultaneously delivering Cas9 protein and sg RNA was prepared for gene editing.The delivery system was assembled by three components,including DNA-g-PCL,DNA linkers,and the Cas9/sg RNA complexes.As the framework of nanogel,the water-soluble DNA-g-PCL furnished multivalent crosslinking sites for nanogel formation.The sequence of brushed DNA was designed to be complementary to the tail of sg RNA that is exposed out from Cas9/sg RNA complex.By partially occupied with Cas9/sg RNA complexes,the remaining DNA brushes on DNA-g-PCL could still be crosslinked by DNA linkers to form nanogel where the Cas9/sg RNA complexes were fully embedded inside.We evaluated the stability of the Cas9/sg RNA-embedded nucleic acid nanogel,and the results demonstrated that the Cas9/sg RNA complexes can be completely embedded inside nanogel,thus resulting in the enhanced tolerance of sg RNA to nuclease.We further evaluated the cellular uptake and gene editing efficiency of nucleic acid nanogel in vitro,and it was demonstrated that the crosslinked nucleic acid nanogel could be rapidly internalized by cells,and the Cas9/sg RNA complexes could be sustainedly released from nanogel,thus inducing that the nanogel delivery system realized efficient target gene editing in vitro.3.The polydopamine-coated nucleic acid nanogel for siRNA-mediated low-temperature photothermal therapyPhotothermal therapy(PTT),as an effective tumor ablation strategy,commonly need to keep the temperature of tumor lesions at?50 ~oC,nevertheless,potentially inducing inflammation,metastasis.Thus,it is vital to achieve effective antitumor effect at relatively low temperature(42-45 ~oC)for PTT clinical transformation.Based on siRNA-crosslinked nanogel self-assembled through nucleic acid hybridization,we designed polydopamine-coated,siRNA-crosslinked nucleic acid nanogel to effective delivery siRNA for low-temperature PTT.The polydopamine,coated on the surface of nucleic acid nanogel via self-polymerization of dopamine in situ,not only supplied the photothermal capacity,but also further strengthened physiological stability and resistance to RNase degradation.We first evaluated the cellular uptake and gene silencing of polydopamine-coated nucleic acid nanogel in vitro,and it was demonstrated that the polydopamine-coated nucleic acid nanogel could be rapidly internalized by cells and realized lysosome escape triggered by laser irradiation.The efficiency of silencing aroused by siRNA-embedded polydopamine-coated nanogel was better than that of Lipofectamine 2000 in vitro.We further assessed the low-temperature photothermal therapy of polydopamine-coated nucleic acid nanogels in vitro.The results revealed the polydopamine-coated nucleic acid nanogel could inhibit the expression of Hsp70 and then achieved the effective low-temperature photothermal therapy.Finally,we evaluated the antitumor effect and pharmacokinetic behavior of polydopamine-coated nanogel,and the results revealed that the polydopamine-coated nanogel had longer blood half-life than that of nanogel,and it could efficiently inhibit the Hsp70 expression during mild near infrared-triggering PTT,resulting in the efficient siRNA-mediated,low-temperature PTT in vivo.