| Background:siRNA delivery remains highly challenging because of its hydrophilic and anionic nature and its sensitivity to nuclease degradation. Effective siRNA loading and improved transfection efficiency into cells represents a key problem. Objective:To prepare Chitosan/Hyaluronic acid-siRNA multilayer films through layer-by-layer self-assembly, in which siRNAs can be effectively loaded and protected. And to explore the stability of a siRNA delivery system and make evaluation on its gene silencing effect.Methods:Chitosan-Hyaluronic acid-siRNA multilayer films were prepared through layer-by-layer self-assembly. Both the construction and the disassembly process of siRNA-loaded films were verified by the UV-vis spectroscopy. The construction process was characterized by FTIR,13C-NMR (CP/MAS), UV-vis spectroscopy, and atomic force microscopy (AFM). We presented the controlled-release performance of the films during incubation in different solution, such as NaCl solution, pH 7.4 PBS solution, and pure water, for several days through UV-vis spectroscopy. (CHS/HA)4(CHS/HA-siRNA)9 films were disassembled by incubation in 1 M NaCl for 6 or 10 days and the released content were collected for PAGE test for verifing the stability and integrity of the siRNA loaded on multilayer films. Finally, the biological efficacy of the siRNA delivery system was evaluated via cells adhesion and gene silencing analyses in eGFP-HEK 293T cells.Results:(1) UV-vis test showed four findings:Firstly, The maximum absorption peak at 260 nm of siRNA-loaded multilayer films showed increased absorbance value with the increasing of the siRNA-layer number, confirmed the LBL deposition. Secondly, the substrates whether dry or not, and the filter processing for the polyelectrolyte solution, could affect the siRNA absorption quantity in multilayer films. And the not dry substrates and filtered polyelectrolyte solution in the LBL process could decrease the siRNA absorption quantity. Thirdly, the salt ion concentration is the important factors that impacted the construction and disassembly process of LBL films in this experiment. And when the salt ion concentration is less than 1 M, the increased salt ion concentration showed much more siRNA loading quantity. Too low or too high salt concentration has no advantage. Fourthly, the siRNA-loaded films showed the better disassembly performance in NaCl of 1 M than in PBS buffer of pH 7.4, and the best stability in pure water incubation.(2) The data from FTIR and 13C-NMR (CP/MAS) spectrum confirmed that both the-NH2 in CHS and -OH in HA participated in the drive assembly and suggested that besides the electrostatic interaction, other interaction such as hydrogen bonding and covalent binding might have been utilized to fabricate this LBL self-assembly. AFM showed the gradually smooth surface of thin films with the increasing of siRNA-loaded layer number. All the appearance of initially hill-shaped protrusions resulting from small particulate matter and subsequent smoothing out surface can verify the multilayered films growth.(3) The UV-vis spectroscopy and polyacrylamide gel electrophoresis (PAGE) experiments showed that the (CHS/HA)4(CHS/HA-siRNA)9 films on the substrates were dissociated into siRNA-loaded complexes first in the incubation of 1 M NaCl of 6 days, and then released free integral siRNA in the incubation of 1 M NaCl of 10 days which was attributed to the appeared single band the same with the physical mixture of HA-siRNA.(4) In the cell transfection experiments, there were three findings as follow:Firstly, both 2-eGFP-siRNA and 7-eGFP-siRNA multilayer films showed a good HEK-293T cells adherence and a gradually decreasing trend (approximately from 80% to 25% in the group of 2-eGFP-siRNA multilayer films) for eGFP gene expression, thus verifing the good eGFP gene sliencing effect. Secondly, with the cells transaction time increasing from 1 to 3 days, the gene silencing effect in 2-eGFP-siRNA group reached best, suggesting that the level of eGFP-HEK 293T cells expressing green fluorescence is likely in a so called dose-dependent manner for eGFP-siRNA, and the release of eGFP-siRNA from 2-eGFP-siRNA films needs some culture time to up to an optimal concentration which could generate high gene silencing efficiency. Additionally, the 7-eGFP-siRNA group showed a better gene silencing effect than in the 2-eGFP-siRNA group, verifing that the gene silencing effect may be associated with the loaded number of eGFP-siRNA layer.Conclusion:A kind of virus siRNA delivery system was successfully constructed, which could open up a feasible way for local siRNA delivery in the near future. |
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