The Construction And Evaluation Of Nucleic Acid Drug Loaded Nanocomplex

Objective:In recent years,gene therapy developes very rapidly.RNA interference(RNAi)is considered as one of the most important discoveries in the treatment of tumor and viral infections.In spite of the potency of siRNA,the application of this kind of nucleic acid are limited by it’s physical and chemical properties including poorer stability,degradability by organisms nuclease,hydrophilicity,electronegativity,and large molecular size.In the studies on the application of siRNA,one of the most important purposes is to enable siRNA to successfully penetrate the cell membrane and approach the cytoplasm.Accordingly,cell-penetrating peptides(CPPs)has attracted much attention.As an amphiprotic or positive short peptide composed of 5-30 amino acids,CPPs can not only efficiently penetrate the cell membrane,but also mediate the translocation of biological macromolecules such as siRNA into cytoplasm.In this study,we chose octahistidine(H8)as the material for binding siRNA.In order to improve the penetrating efficiency of H8,we conjugated H8 with hydrophobic matter(stearic acid,SA),followed by the preparation of SA-H8、SA-H8-PEG2000 and the mixture of these two positive carriers.Based on electrostatic interactions,siRNA was condensed by positive carriers such as SA-H8-PEG2000/siRNA and SA-H8-PEG2000n/siRNA nanocomplex.By measuring the potential,particle size of nanocomplex and the agarose gel experiment(AGE),we investigated the physical and chemical properties of SA-H8-PEG2000/siRNA and SA-H8-PEG2000n/siRNA and optimized the formulation.We analyzed the interaction between complexed siRNA and MCF-7/A549 cells,and envaluated the uptake efficiency of cargo.Methods:On the basis of the documents and experiments,we optimized the synthesis routes and purification method of SA-H8 and SA-H8-PEG2000.We mixed the SA and H8 at room temperature,and stirred the mixture about one hour,followed by the dialysis and freeze drying.After the formation of SA-H8,we conducted the conjugation of SA-H8 with mPEG2000-Mal by stirring the mixed system at room temperature.In view of the particle size,potential and gel retardation,we characterized the physical and chemical properties of nanocomplex,and screened out the qualified formulation.We chose the MCF-7 cells and A549 cells as cell models and evaluated cellular uptake efficiency of the complexed siRNA depending on the analysis of fluorescence intensity by flow cytometry.Results:Mass spectrometry assay demonstrated that both SA-H8 and SA-H8-PEG2000 were synthesized successfully.Following the analysis of zeta potential,particle size and gel retardation ability,SA-H8-PEG200020%/siRNA nanocomplex have particle size between 200 nm to 700 nm,and a weak compressibility of siRNA.In order to improve the condensation of siRNA,we considered increasing the proportion of SA-H8-PEG2000 in SA-H8-PEG2000 n.SA-H8-PEG200050%/siRNA nanocomplex have particle size between 350 nm to 450 nm,and siRNA suffered from full loading when N/P ratio went up to 80:1.Also,SA-H8-PEG2000/siRNA nanocomplex have particle size between 300 nm to 400 nm,and completely encapsulated siRNA once N/P ratio increased to 40 or more.Above all,we selected the SA-H8-PEG200050%/siRNA 80:1 and SA-H8-PEG2000/siRNA 80:1 as the final formulations.Flow cytometry analysis demonstrated that both SA-H8-PEG2000 /siRNA and SA-H8-PEG200050%/siRNA offered elevated cellular uptake compared with free siRNA,while their internalization was much lower than that of commercially available products.Conclusions:In this study,the carriers effectively loaded siRNA and significantly improved it’s cellular uptake.However,in contrast to transfection ability of commercial products,it is necessary to continue to optimize the formulation and preparation process of the carrier.