Zwitterionic PH/Redox Sensitive Nanoparticles For Pulmonary Delivery Of SiRNA

Objective:In order to establish a novel zwitterionic pH and redox sensitive nanoparticles containing STAT3-siRNA for pulmonary administration,we synthesized Dextran-Succinic anhydride-Cystamine(DeSC)followed by preparation and evaluation of DeSC nanoparticles in vitro and in vivo.Methods:Cross-linked polymer Dextran-Succinic anhydride-Cystamine(DeSC)was synthesized by esterification and amidation reaction,and the structure was validated by 1H-NMR and FT-IR.The pH and redox dual-sensitive nanoparticles(DeSC-NPs)were prepared by self-assembly method.Various PEI/siRNA complexes were loaded in the nanoparticles(siRNA@DeSC-NPs)by electrostatic interaction.Gel retardation assay was conducted to optimize the formulation of nanoparticles.Particle size and zeta potential assays were performed to characterize the DeSC-NPs.The tests of pH and redox sensitivity of DeSC-NPs were also carried out.The protective effect of DeSC-NPs on siRNA and in vitro release in different buffer solution was investigated.Safety of polymer and nanoparticles on A549 cells and 293T cells were observed using MTT assay.Flow cytometry(FCM)and laser confocal microscopy(CLSM)were used to evaluate the uptake and intracellular distribution of siRNA@DeSC-NPs in different pH conditions.The apoptosis assay and anti-proliferation ability of STAT3-siRNA@DeSC-NPs on A549 cells were investigated using flow cytometry(FCM)and MTT method,respectively.The gene silencing effect of STAT3 mRNA was measured by RT-qPCR and the protein silencing effect of STAT3 was examined by Western Blot.Orthotopic lung tumor model in nude mice were established by intrathoracic injection of Luciferase-A549 cells.The distribution of Cy5-siRNA@DeSC-NPs and the inhibition of tumor growth of STAT3-siRNA@DeSC-NPs by tail vein injection and pulmonary inhalation in tumor bearing nude mice using animal imaging system.Results:The structure of DeSC was confirmed by 1H-NMR and FT-IR.The polymer showed low cytotoxicity and good biocompatibility.The siRNA@DeSC-NPs had a uniform particle size distribution and charge-reversal features depending on the environment pH condition.The gel retardation assay indicated that DeSC-NPs had high encapsulation efficiency and protection effect of siRNA.In vitro release results revealed that the DeSC-NPs can efficiently release free siRNA in the simulated tumor environment.The cellular uptake and intracellular distribution indicated that the uptake of nanoparticles by A549 cells was time-dependent and pH-dependent.CLSM showed that siRNA in the nanoparticles effectively escaped from the lysosomes by the proton sponge effect caused by PEI and DeSC.The apoptosis ratio and the anti-proliferation effect were promoted by STAT3-siRNA@DeSC-NPs.RT-qPCR and Western Blot assay indicated that STAT3-siRNA@DeSC-NPs could effectively silence STAT3 mRNA and STAT3 protein.The in vivo study showed that STAT3-siRNA@DeSC-NPs could be accumulated in the lungs of nude mice by both of tail vein injection and pulmonary inhalation,which effectively inhibited tumor growth.The results of H&E staining confirmed low toxicity of DeSC-NPs.Conclusions:A novel pH and redox dual-sensitive nanoparticles containing STAT3-siRNA was designed and evaluated.The nanoparticles showed robust potential as siRNA carrier in present study.The futher investigation is under going.