Protein-controlled Synthesis Of Chitosan Nanoparticles For Efficient SiRNA Transfection And Fluorescence Imaging

RNA interference (RNAi) is a biological process in which RNA moleculesinhibit gene expression, typically by causing the destruction of specific mRNAmolecules. siRNA involved in RNAi process, inhibits the expression of specific genes,and is a hot issue for gene therapy. However, siRNA delivery remains highlychallenging. This is in part attributable to the nature of siRNA, which is sensitive tonuclease degradation. Moreover, siRNA is hydrophilic and anionic and is unable tocross the cellular membranes. Many previous studies in siRNA delivery achievedmuch progress, but there are limitations in applications. For example, viral vectorshave a high transfection efficiency, but the safety of virus, the high expense and thecomplicated precedure in viral vector preparation hamper its applicability. Thecommonly used cationic liposome carrier has the cell toxicity and instability. PEI andCNTs are also widely used for siRNA delivery, but the safety of them have beenhighly controversial. So it is important for us to explore the carriers with safety, highefficiency, and stability.Chitosan-based nanoparticles (NPs) have been widely described investigated inthe delivery of nucleic acids such as siRNA or DNA because of its cationic nature,low toxicity, biocompatibility, and biodegradability. One of the common methods ofnanoparticles formulation was coprecipitation, but the biggest weakness of NPssynthesed by this method is the poor stability in the transfection medium, and the lowfinal silencing efficiency. By adding a cross-linker agent in the formulation, such assodium tripolyphosphate (TPP), the interactions between siRNA and the polymer arestrengthened. This approach, called ionic gelation, permits higher NPs stability.However, the NPs generally have a large size and uniform size distribution, and causethe poor dispersion and stability.An efficient chitosan-based siRNA delivery nano vector, P@CS, has beensynthesized. Using proteins with different sizes and surface charges as the nucleationcores, the size of P@CS NPs have been tuned into around20nm, which is regardedas the optimal size for siRNA delivery. Originated from the size and charge- distribution of the protein cores, P@CS has a non-spherical shape, which provideshigh binding capacity for siRNA.Confocal fluorescence microscopy and flow cytometry experiments show thatP@CS NPs deliver siRNA into cells efficiently. Real-time PCR and Western-blotexperiments exhibit that P@CS NPs delivered siRNA silences GAPDH gene with anefficacy much higher than the commercial lipofectamine2000. Notably, some of thesiRNA are even delivered into the cell nuclei, where they may intercept the targetmRNA before it translocates from the nuclei to the cytoplasm, thus accounts partly forthe high gene silencing effect. Due to the good biocompatibility and biodegradabilityof both protein templates and chitosan, P@CS NPs are non-toxic to both HeLa andCaco-2cells at a dose up to400μg/mL. The good transfection efficacy, the ability todeliver nucleic acids into cell nuclei and excellent safety demonstrate the promisingpotentials of P@CS NPs for the gene therapy.We choose the RFP as the core to synthesis the PR@CS NPs to explore the newbiological fluorescence probe for imaging. It is found that the chitosan encapsulationdid not affect the fluorescence of RFP, and could significantly improve the stabilitiesagaint denaturant, proteinase, and photobleaching. All of these properties showing itspotential application for bio-imaging.RFP was chosen as the core for tracking the siRNA delivery by fluorescence. Theimproved stability and fluorescence performance demonstrate that PR@CS is a goodprobe to monitor the delivery of siRNA to cells. So we prepared the multi-functionalnanocarriers for siRNA delivery and imaging at the same time. It has the promisingpotentials for the gene therapy and fluorescence imaging.