Active Targeting And Hierarchical Regulation Strategies For Construction Of Nucleic Acids Delivery Systems

Sequential physiological obstacles encountered in the journey of intracellular gene delivery substantially hindered the therapeutic efficiency of gene therapy.Development of appropriate gene delivery vehicles capable of resolving the pre-defined these physiological obstacles has motivated intensive interests of the researchers in the field of gene therapy.In the present research,a multi-responsive nanostructure composed of block copolymer FA-PEG-GPLGVRG-P[Asp(DET)]was designed and tailored to possess active targeting and hierarchical regulation functionalities.The proposed strategies are envisioned to benefit the delivery efficiency of the following critical delivery stages:including blood circulation,tumor accumulation,tissue penetration,cell internalization and drug release through ingenious chemistry design.1.Synthesis and characterizations of FA-PEG-GPLGVRG-P[Asp(DET)]Gene delivery systems were synthesized by clicking reaction,ring opening polymerization and substitution reaction,with FA as the polit molecule,PEG as the hydrophilic block,and P[Asp(DET)]as the nucleic acids-loading block.The chemical structure of the polymer was characterized by FTIR and ~1H NMR,The results verified that the proposed block copolymer of FA-PEG-GPLGVRG-P[Asp(DET)]was successfully synthesized and the polymerization degree of P[Asp(DET)]was approximate 70.2.The physicochemical properties and biological performance study of F-G-P/pDNAThe delivery systems and pDNA are self-assembled into polyplex by electrostatic interaction.DLS and TEM results indicated that polyplex F-G-P/pDNA possess nanoscaled spherical structure with uniform distributed,the particle size approximate 80 nm and zeta potential approaching electric neutrality.These results indicated the condensing ability of FA-PEG-GPLGVRG-P[Asp(DET)]to pDNA and its potential in circulation in the bloodstream.Gel retardation assays have confirmed that FA-PEG-GPLGVRG-P[Asp(DET)]can effectively bind to pDNA through electrostatic interaction,neutralize the negative charge of pDNA,conferring protection of genomic payloads.The estimation of hemolysis and cytotoxicity observed the hemolysis rate of the constructed gene delivery systems less than5%and the cell survival rate exceeding 85%,implying excellent safety profiles.Moreover,the evaluations of cell uptake and in vitro gene transfection demonstrated appreciable efficiency of the cell uptake efficiency and transfection efficiency from constructed gene delivery systems.3.Performance study of F-G-P/pDNA responsive dePEGylation and two-step protonation in response to intracellular acidic milieuThe membrane disrupting ability,uptake efficiency and endosome(lysosomal)escape ability of the constructed gene delivery systems with and without MMP-2 incubation were qualitatively and quantitatively characterized by LDH experiment,cell uptake experiment and co-localization experiment.The results of LDH experiment showed that the membrane disrupting ability of constructed gene delivery systems incubated with MMP-2 was significantly enhanced,thus significantly elevated the cell uptake efficiency,which was consistent with the results of cell uptake evaluation.In addition,the P[Asp(DET)]block has distinctive pH-responsive character,two-step protonation profile in responsive to the pH gradient upon acidic endosome entrapment,and the membrane disrupting capacity of the constructed gene delivery systems at pH 5.5(acidic endosomes)is significantly greater than that at pH 7.4(physiological pH),thereby affording stronger endosome(lysosomal)escape ability.The results of co-localization experiment also confirm this claim.4.In vivo evaluation of tumor suppressionThe expression of sFlt-1 pDNA was encapsulated into F-G-P-based gene delivery system,and the PBS control group and PEG-GPLGVRG-P[Asp(DET)]/pDNA experimental group without active targeting moiety were included as control.The mouse glioma tumor model was established using U87 glial tumor cells,and tumor suppression evaluation was conducted.The subsequent evaluations verified the potent inhibitory effect of F-G-P/pDNA with folic acid groups,and the mice in this group showed the most retarded tumor growth,with an average survival time of 25 days,which was significantly greater than that in the P-G-P/pDNA experimental group for 19 days and the PBS control group for 12 days.These results demonstrate that tempting active targeting function of folate groups and intriguing potential of gene therapy with F-G-P delivery system.