Preparation Of Positively Charged Fluorescent Conjugated Polymer Dots And Their Applications In Gene Delivery

As the new way to tackle genetic diseases and cancer,gene therapy has been attracted great attention and widely investigated.To find out a high effective and low toxic gene delivery vector is one of the most important missions now.A wonderful gene delivery vector can not only introducing genetic drugs into specific cells,but also maintaining its function.In the past few decades,scientists devoted to seck efficient non-viral gene delivery vector.Fluorescent conjugated polymer nanoparticles(CPNs)have been developed as a bright nanoprobe in biological analysis and imaging,as well as in diagnosis and drug delivery.CPNs are well-suited for these different applications because of exists exceptional photophysical properties,including large extinction coefficients,high single-particle brightness,and excellent photostability.However,there are few reports about CPNs on gene therapy.In order to improve the transfection efficiency and the in vivo stability of non-viral vectors,this thesis used CPNs as carriers for efficient gene delivery.The work was performed as follow:(1)Through nanoprecipitation of fluorescent conjugated polymer,poly[(9,9-dioctylfluorenyl-2,7-diyl)-co-(1,4-benzo-{2,1?,3}-thiadiazole)](i.e.PFBT)with zwitterionic lipid molecules(propylene glycol amine cationic lipid)together,positively charged CPNs were obtained.TheseCPNPs possess decent fluorescence performance,with fluorescence quantum yield of 70.7±0.3% and small size dimension of ~3.6±0.3 nm.(2)As a effective fluorescent nanoprobe,CPNs were widely applied to intracellular fluorescence imaging and efficient gene transfection.According to the living cell imaging experiments,we found that CPNs have fast and efficient cellular translocation capability.Nearly 100%cellular loading efficiency was observed after co-incubation of CPNs with many kinds of cells.In comparison with the commonly used gene delivery vector,lipofectamine 2000,the gene expression efficiency of the positively charged CPNs was improved significantly.Owing to the merits as described above,the CPNs would afford a promising non-viral platform for therapeutic nucleic acids delivery in the future.(3)With high spatial resolution of optical microscopy imaging techniques,this work explored the interaction between the gene delivery nanocarriers and the cell membrane,and time-correlated aggregation and redistribution information inside living cells.The results showed that energy-dependent endocytosis was the prevailing pathway for the cellular uptake of CPNs.Intracellular fluorescence imaging results demonstrated that the CPNs could actively assemble close to the periphery of nuclei.Disassembly was not observed even 36 h later,which greatly facilitates releasing of pDNA close to the periphery of nuclei and thus promotes the gene transfection efficiency.These understandings on the cellular uptakemechanism and intracellular fate of CPNs in living cells would provide deep insight on how to improve gene expression efficiency when use CPNs.