Preparation And Characterization Of Chitosan/siRNA Nanoparticle Carrier And Its Gene Silencing

RNA interference is a natural phenomenon in the organisms: a double-strandedRNA generated by intracellar introduction or endogenous transcription, binds to thehomologous mRNA and mediates the targeted mRNA degradation, which causes thesilencing of the corresponding gene after the transcription. On one hand, RNAitechnology was readily appreciated as a powerful tool in gene phenotype and functionvalidation experiments. For the identification of the disease mechanism, genetargetting therapy, development of new drug such as micromolecule compounds andthe discovery of antibody drugs, RNAi has provided theoretical basis. On the otherhand, based on the previous study, RNAi technology can be used as a drug candidatefor the treatment of human disease. Small interfering RNA(siRNA) is the effectormolecule of the repression function of RNAi. With the dual characteristics of nucleicacid and micromolecule compounds, siRNA molecules are susceptible to nucleaseand have poor stability, short half-life, low transfection efficiency, poor targeting andso on. The key to the application of siRNA molecules, is how to overcome thecellular membrane barrier, and make siRNA molecules into the RNAi pathway incytoplasm. Cationic liposomes, viral vector, some physical techniques such as genegun, electroblot, etc., have so far been used in transfecting siRNAs into cells. However, introducing siRNA molecules into body to treat diseases is much morecomplicated. Although with a high efficiency of gene transfection, viral vector has apotential carcinogenicity and immunogenicity, and cationic liposome has a larger celltoxicity and instability. In recent years, the potential of chitosan as a polycationicgene cartier for siRNA has been explored in several research groups, achieved goodresults in vitro and in vivo experiments.Chitosan,α(1-4)2-amino-2-deoxy-β-D-glucan, is a potential natural cationicpolysaccharide extracted from crustacean shells and obtained by the alkalinedeacetylation of chitin. Chitosan has a wide variety of sources, good biocompatibilityand biodegradability, low toxicity and immunogenicity. Due to its goodbiocompatibility and toxicity profile, it has been widely used in pharmaceuticalresearch and in industry as a carrier for drug delivery and as biomedical material forartificial skin and wound healing bandage applications. In recent years, some peopletry to make chitosan as the delivery system in vitro and in vivo of genes, which haveachieved good results. Chitosan is the only polysaccharide with weak alkaline, atacidic pH, below 5.5 or so, and the primary amines in chitosan become positivelycharged. These protonated amines enable the chitosan to bind to negatively chargednucleic acid molecule and self-induced complex coacervation into mic- ornanoparicles through phase separation. This mic- or nanoparicles can partially protectDNA from nuclease degradation. The particles accumulated by chitosan and nucleicacid can be easily uptaken by reticuloendothelial system. Chitosan nanoparticle canenter intracellular by internalization, be degraded in vivo by enzymes, such aslysozyme and chitosanase, into oligomers and further to N-glucosamine, which isendogenous to the human body, then release the DNA molecule. Mucoadhesive andmucosa permeation properties of chitosan potentially permit a sustained interaction ofthe macromolecule to be "delivered" with the membrane epithelia, promoting more efficient uptake. Therefore, chitosan polymer can effectively solve some keyproblems in the gene transfection process, such as the cell absorption, intracellularrelease, nucleic location and so on, which has a good prospect as a gene deliveryvector.In order to explore the feasibility of chitosan as siRNA vector, with a complexcoacervation progress we prepared a kind of chitosan nanoparticle which couldencapsulate both plasmid DNA and siRNA molecules in this study. The structuralfeatures and gene transfection efficiency in vitro of this chitosan nanoparticle withDNA plasmid or siRNA molecules have been characterized. The gene silencingefficiency of chitosan-siRNA nanoparticle has been investigated, which lay thefoundation for further application in vivo.Objectives:1. To study the potention of chitosan nanoparticle as a polycationic siRNA deliverysystem for in vitro RNA interference application.2. To prepare chitosan nanoparticles carrying pGFP and study its pharmaceuticalcharacteristics and gene transfection efficiency in vitro; and to demonstrate thepotential of chitosan as a gene carrier for nucleic acid biomolecules3. To prepare chitosan-siRNA nanoparticles and clear whether the siRNA moleculescould be transfected into the cultured cells; and to reason the application ofchitosan as a gene carrier for siRNA moleculars.4. To study whether the siFHL2 molecule could be transfected into the cultured cellsefficiently and the targeting gene FHL2 could be silenced by this chitosannanoparticle.Methods: 1. The plasmid expressing green fluorescent protein (pGFP) was used as report gene.Chitosan-pGFP nanoparticles were prepared using a complex coacervationprocess. The binging of pDNA in chitosan nanoparicle was speculated by 1.0%agarose gel electrophoresis analysis. The encapsulating rates were determined bycolorimetry. The size distribution and polydispersity of nanoparicles weremeasured by nanoparticle size analyzer; the morphology was observed by atomicforce microscope and scanning electron microscope.2. To investigate the transfection efficiency of Chitosan -pGFP nanoparticles in thehuman colon adenocarcinoma cell line LoVo in vitro, with Lipofectamine 2000gene carrier as positive control, the cell population expression of greenfluorescence protein have been detected by fluorescence microscopy.3. The control siRNA molecules were labeled by FAM. The chitosan-control siRNAnanoparticles were prepared by complex coacervation progress. 24 hrs aftertransfection of this nanoparicle, the green fluorescent in LoVo cells was examinedwith laser scanning confocal microscopy, and the transfection efficiency and theintracellular distribution of siRNAs have also been investigated.4. The chitosan-siFHL2(siRNA targeted to FHL2 mRNA) nanoparticles wereprepared by the said complex coacervation progress. The total mRNA of LoVocells was extracted by Trizol reagent and the FHL2 gene expression wasmeasured by semi-quantitative RT-PCR.The sequences of siFHL2 were as follows:sense: 5'-CGAAUCUCUCUUUGGCAAGdTdT-3';antisense: 5'-dTdTGCUUAGAGAGAAACCGUUC-3'The PCR primers for control GAPDH were as follows:sense sequence(GF): 5'-AAGGTCATCCCAGAGCTGAA-3';antisense sequence(GR): 5'-ATGTAGGCCATGAGGTCCAC-3'. The PCR primers for FHL-2 were as follows:sense sequence(FF): 5'-ATGACTGAGCGCTTTGACTG-3';antisense sequence(FR): 5'-AGTTCAGGCAGTAGGCAAAGTC-3'.Results:1. Chitosan-pGFP nanoparitcle was obtained by a complex coacervation process. Thegel electrophoresis showed most of pGFP was to be detained in the well, whichindicated that chitosan could almost completely combine the pDNA by electroniccombination. The encapsulating rates for these nanoparticles were all more than90%. The morphology of most chitosan nanoparticle was spherical. The meandiameter of this nanoparticle was about 209 nm with polydispersity of 0.15.Lyophilized nanoparticles have irregular shape of lamellar or dendritic observedby scanning electron microscopy. Resuspension of this nanoprticle by ethanol, theshape changed into spindle particles with different sizes.2. The transfection of chitosan-pGFP nanoparticle in vitro was efficient and theexpression of green fluorescent proteins was observed by fluorescentmicroscope, which indicated that chitosan can deliver pGFP into LoVo cells.However, the transfection efficiency of chitosan nanoparticle was lower than thatof liposome.3. Chitosan-control siRNA nanoparitcle was obtained by a complex coacervationprocess. After transfecfion of these nanoparticles into LoVo cells, the majority ofcells had green fluorescence observed under laser scanning confocal microscopy.These green fluorescent were mainly distributed in cytoplasm, along the internalside of cell membrane and the circle of nuclear membrane. The same distributionof green fluorescent was obtained in Liposomes/siRNA transfection complexesgroup, and there wasn't any green fluorescent in the negative control group. The results here indicated that both liposome complexes and chitosan nanoparticlescould delivery the siRNA molecules into LoVo cells.4. Chitosan-siRNA nanoparitcle was obtained by a complex coacervation process.The gene silencing of siFHL2 molecules mediated by chitosan nanoparticle wasefficiency. The expression level of FHL2 gene was down regulated. There wassignificant difference on the expression of FHL2 mRNA among the four groupsby one-way ANOVA analysis (F=1138.441, P＜0.001). Further statisticalanalysis(by LSD) indicated that the FHL2 mRNA levels between the groups ofliposome-mediated and chitosan nanoparticle-mediated transfection of siFHL2molecules were no significant difference(P=0.219), and lower than that innon-transfected control group(P＜0.001), and also lower than that in controlsiRNA-chitosan nanoparticle-transfected group(P＜0.001). The FHL2 mRNAlevels between control siRNA chitosan nanoparticle-transfected group andnon-transfected control group was no significant difference (P=-0.298).Conclusions:1. Chitosan nanoparticle prepared by complex coacervation could combine thepDNA efficiently with high encapsulating rate and the diameter distribution was100～500nm. Nanoparticles were aggregated in the lyophillization process thatleads to a greater change of shape and diameter.2. Chitosan nanoparticles can deliver plasmid DNA into cells in vitro, and the reportgene can be expressed in cells.3. SiRNA molecules can be delivered into cells by chitosan nanoparticles in vitro,and can induce RNAi effect on the targeting gene. Therefore the preparedchitosan nanoparticle has a prospect to be an effective delivery system for siRNAmolecules.