Stuides On A Micelleplex Delivery System Based On Dual Responsive-ness And Hydrophobic Force

Despite the extracellular barriers for siRNA delivery have been overcome by utilizing advanced nanoparticle delivery systems,the key intracellular barriers after internalization including efficient disassembly of siRNA and endosomal escape still remains challenging,especially for systematic siRNA delivery.The main objectives of the study were to developed a unique pH-and redox potential-responsive polyplex delivery system based on the copolymer of mPEG-b-PLA-PHis-ssPEI1.8 k,which is composed of a pH-responsive copolymer of PEG-b-PLA-PHis(Mw 5 k)and a branched PEI(Mw1.8 k)linked with redox cleavable disulfide bond.The polyplexes were designed to effectively disassembly of free siRNA in endosome by reducing enzyme(GILT)γ-interferon-inducible lysosomal thiol reductase in combination with cysteine followed by fast ensodomal escape in order to combat intracellular barriers.The polyplexes were expected to fulfill several sequential tasks:1)complexing siRNA and self-assembling into PEG shielded polyplexes by integrating the hydrophobic force of PLA-PHis,electrostatic attractions of cationic PEI and hydration effect of PEG,2)precisely responding to the acidity and reduction potential in endosome and triggering the efficient disassembly of siRNA due to the structure change of the polyplexes,3)facilitating efficient endosomal escape of siRNA via the endosomal membrane destabilization of the detached PEI as well as the proton sponge effects.Firstly,the stimuli-responsive copolymer mPEG-PLA-PHis-ssPEI 1.8k was sythesized and characterized by FT-IR,1H-NMR,MS and GPC.The pKa value of the copolymer measured by titration was 6.8.The copolymer showed good buffer capacity in the pH range of 4.0～7.4 in the titration curve.The hemolysis of copolymer was investigated with rabbit erythrocyte suspension,which indicated that mPPP-ssPEI was in lower hemolysis and better safety,compared with PEI.The micelleplexs in different N/P ratios were investigated by agrose gel electrophoresis,fluorescence spectrophotometry and dynamic light scattering.The results showed the copolymer cound bind siRNA and assemble into intact micelleplexes with encapsulation efficiency over 89%,when N/P ratio is above 3.The particle size and zeta potential increased depending on N/P ratio.The micelleplexes were able to resist replacement by heparin with N/P ratio above 4 and protect siRNA from degradation by nuclease in the serum with N/P above 6.The responsive release of siRNA from micelleplexes was determined by agrose gel electrophoresis and fluorescence spectrophotometry and Transmission Electron Microscopy.After incubated at pH 5.5 in the presence of 10 mM DTT,the structure of micellelplexes became not intact,and the some tiny gaps appeared in morphology.Meanwhile,there was a great increase in particle size,indicating the redox-triggered structure change of polyplexes.Obvious accelerated siRNA release from the polyplexes was observed at N/P ratio below 8 after treated with pH 5.5 and 10 mM DTT.The pH and DTT triggered siRNA release from the polyplexes was markedly restrained when the N/P ratio was raised above 10.The DTT accelerated the disassembly of siRNA from the polyplexes was dependent on the pH decrease.The DTT contribution to the siRNA disassembly from the polyplexes was negligible at pH 7.4.However,the DTT contribution was markedly increased when the pH decreased to 5.5,mainly because the pH induced structure change of polyplexes contributed the siRNA disassembly by exposing more disulfide bonds in the reductive environment for cleavage.The MTT assay showed that different micelleplexes showed more than 80%cell viability over 72 h in all three cell lines,indicating the good biocompatibility and low cytotoxicity of the micelleplexes.By confocal laser scanning microscopy,three micelleplexes treated cells showed more dispersive and broader distribution of fluorescence signals and lower lysotracker colocalization in the cells than Lipo2000 treated cells.This suggests that the polyplexes are able to efficiently facilitate the endosomal escape of siRNA after its disassembly.after the shut-off ATPase proton pump with bafilomycin A1,the polyplexes(N/P 6)treated cells showed comparable Cy3-siRNA dispersive distribution but only slightly less lysotracker colocalization than the cells with normal ATPase proton pump,indicating that“proton sponge effect”might not work as efficiently as free PEI segments in facilitating siRNA endosomal escape.By fluorescence resonance energy transfer(FRET)assay,the FRET efficiency of polyplexes(N/P 6)was significantly lower than polyplexes(N/P 10)and polyplexes(N/P 15)respectively,indicating a higher disassembly extent.The protein downregulation level of micelleplexes were investigated by western blotting and luminescence assays,which showed that the protein downregulation of polyplexes at N/P ratio of 6 was significantly higher than polyplexes with higher N/P ratios at 10 and 15,indicating that the polyplexes(N/P 10 and 15)did not disassemble to release siRNA as efficiently as polyplexes(N/P 6)in the endosome because of the stronger electrostatic binding inside the complexes,leading to lower transfection efficiency.Besides,the gene silencing of the polyplexes(N/P 15)is higher than that of polyplexes(N/P 10),mainly because both of the polyplexes showed similar siRNA release profile,however micelleplexes with higher N/P ratio,produced the more efficient endosomal escape of siRNA.The biodistribution of polyplex delivery system in vivo was examined using Cy5-labeled siRNA through subcutaneous injection into MCF-7 xenograft nude mice.The results showed that the polyplexes has relatively long circulation,fast accumulation in tumor and gradual elimination through kidney compared to naked siRNA,and three micelleplexes did not exhibit significant difference.In vivo antitumor effect of polyplexes was investigated using a breast malignant tumor model established through inoculating MCF-7 cells into the female BALB/c mice.Three micelleplexes inhibited the tumor growth significantly compared to saline and scrambled siBcl-2 loaded polyplexes.The polyplexes(N/P 6)demonstrated better antitumor effect and Bcl-2 downregulation than the polyplexes(N/P 10 and N/P 15),verifying that the siRNA responsive release of micelleplexes contributed most to the efficient tumor growth inhibition and protein downregulation.