| Objective:In recent years,RNAi has received more and more attention in term of gene function and gene therapy.And it has gradually developed into a mature technology.RNA interference is a specific cellular post-transcriptional gene silencing mechanism,which can be induced by evocation of enzymatic degradation of a corresponding mRNA.Double-stranded small interfering RNA(siRNA)plays an important role in this process.siRNA can be synthesized exogenously and delivered to the cytoplasm,where it induces AGO(argonaute)protein to cleave the mRNA and interrupts the translation process.As a kind of potent and highly specific therapeutic agent,siRNA has been applied to treat a wide range of gene-based diseases.However,the major limitations for the use of siRNA are the instability of naked siRNA in physiological conditions,the nature of negative charge,and hydrophilicity.Currently,various strategies have been utilized to improve siRNA’s nuclease resistance without interfering with its silencing efficiency.Based on the high efficiency,low toxicity and so on,polypeptide carriers have been a hotspot.Among them,cell-penetrating peptides(CPPs)are a type of short peptides that can mediate the translocation of macromolecular substances across cell membrane,and have received considerable attention.In this paper,octa-arginine(R8)was selected as the carrier material.It was modified by stearic acid and polyethylene glycol via covalent bond.So several functional polymers were synthesized,which can be assembled into nanoparticle spontaneously.Then using these polymers to condense siRNA,several nanocomplexes was prepared.A series of experiments were conducted to investigate the basic properties of nanocomplex.The nanocomplex which was capable of binding,protecting and delivering siRNA into cells wasscreened out.Finally,MCF-7 and A549 cells were used to investigate the cellular uptake of the optimal complex loading siRNA.Methods:1 Conjugation.First of all,SA and R8 was conjugated through the amide bond.The SA reaction solution was prepared firstly,then the SA solution reacted with R8 solution for 30 min.After that,the SA-R8 reaction solution was dialyzed by purified water for 24 h.Finally,the dialysate was detected by mass spectrometry analysis.Then SA-R8 was reacted with mPEG2000-Mal or mPEG5000-Mal to synthesize SA-R8-PEG.2 Preparation and evaluation of nanocomplex.Due to amphipathy,SA-R8 can be assembled into nanoparticle spontaneously in the polar solvent.R8 has positive charges,so SA-R8 and SA-R8-PEG can condense negatively charged siRNA.Based on the principle,several siRNA-loaded nanocomplexes was prepared in DEPC solution.Then the particle size and zeta potential of different N/P ratios nanocomplexes was measured through the nano-particle size and zeta potential analyzer;the agarose gel electrophoresis was performed to explore the nanocomplex’s ability of packaging and deliverinng siRNA into tumor cells.Finally,using MCF-7 and A549 as the model cells,the cellular uptake effect of the optimal formulation was evaluated by flow cytometry.Results:The mass spectrometry showed that the practical molecular weight of SA-R8,SA-R8-PEG2000 and SA-R8-PEG5000 was consistent with the theoretical data,respectively.The average particle size of(SA-R8/SA-R8-PEG200020%)/siRNA was between 300~400nm,and(SA-R8/SA-R8-PEG500020%)/siRNA’ average particle size was between 500~800nm;with the N/P ratio increasing,the surface charge of both(SA-R8/SA-R8-PEG200020%)/siRNA and(SA-R8/SA-R8-PEG500020%)/siRNA changed from the negative charge to the positive charge.The agarose gel electrophoresis test showed that both(SA-R8/SA-R8-PEG200020%)/si RNA and(SA-R8/SA-R8-PEG500020%)/siRNA can condense siRNA completely at the N/P ratio of 20:1.In vitroessay showed that two types of siRNA-loaded nanocomplexes were able to successfully transfect siRNA into tumor cells,part of which was comparable to the level of commercially available product.Conclusions:1 Carrier materials were successfully synthesized.The method was simple and reproducible.2 siRNA-loaded nanocomplexes were successfully prepared,and the method was also simple and reproducible.The characteristics of particle size and zeta potential proved that the nanocomplexes were relatively stable;and the agarose gel electrophoresis test proved that the nanocomplexes could package the siRNA.3 Cellular uptake assay showed that both(SA-R8/SA-R8-PEG200020%)/siRNA and(SA-R8/SA-R8-PEG500020%)/siRNA significantly improved the cellular uptake of siRNA at the N/P rato of 20:1,compared to free siRNA.There was no significant difference(P>0.05)in the transfection of siRNA into cancer cells between the SR/SRP2 K group and the commercial product.In the future,we will continue to optimize the formula and preparation technique of the delivery system to promote its transfection efficiency,and provide a new strategy for tumor therapy. |
