| Gene therapy,the process to deliver therapeutic nucleic acids into targeted cells to correct genetic disorders,becomes a promising treatment for genetic diseases such as cancers,which is featured by its high efficacy and low toxicity.The bottleneck to its clinical applications is the lack of safe and efficient vectors.For in vivo gene delivery,the vectors can be categorized into two classes:viral vectors and non-viral vectors.Viral vectors,the engineered viruses carrying genes,can achieve high transfection efficiency and sustained gene expression.However,their applications in clinic trials are limited by the biosafety concerns and difficulty in large-scale preparation.Non-viral vectors,especially the cationic polymers,are attractive candidates due to their low immunogenicity and toxicity,as well as the potential for scale-up manufacture.However,the primary drawback of cationic polymers is their decreased transfection efficiency by serum.For example,the transfection efficiency of branched polyethylenemine of 25 kDa molecular weight(bPEI25k),the gold standard for in vitro transfection,can decrease by 2-3 orders of magnitudes in the presence of serum.The current mechanism studies about hpw serum proteins affect polymer/DNA complex(polyplex)mediated transfection remain controversial,and no comprehensive and systematic study has been reported so far.Therefore,in the second chapter,we investigated the effects of serum proteins or bovine serum albumin(BSA)on physicochemical properties and transfection of bPEI25k polyplexes.Serum proteins and BSA were adsorbed on the surface of bPEI25k polyplexes to form the stable ternary polyplex/protein core-shell structures(herein,both PEI/DNA polyplexes and PEI/DNA/protein are referred to as polyplexes).Their physicochemical properties including sizes,zeta potentials,morphologies and salt sensitivity were evaluated.Three representative PEI/DNA/BS A polyplexes at weight ratios of 1/1/0(PEI/DNA),1/1/4 and 1/1/40 were further selected to study their transfection and related mechanisms.The results showed that protein corona determined the endocytic rates and pathways,the rate of endo/lysosomes transporting,vesicle escape efficiency and gene expression levels.PEI/DNA and 1/1/4 polyplexes entered cells via non-digestive pathways,namely caveola-mediated endocytosis or macropinocytosis respectively,exhibiting high transfection efficiency.1/1/40 polyplexes were rapidly transported into lysosomes via clathrin-mediated endocytosis.Their poor vesicle escape efficiency and the subsequent degradation resulted in extremely low gene expression.In the third chapter,we further investigated the gene expression of PEI/DNA and 1/1/4 polyplexes on the overall and single cell levels.On the overall level,we compared the gene expression rates of two polyplexes at timed incubation and transfection,the effects of DNA concentrations on gene expression levels(including expressing cell ratios and expressing levels)and the difference in two plasmids' co-transfection.On the single cell level,we evaluated the mitosis-dependent gene expression,the time from cell mitosis to their expression(onset time)and single cell gene expression patterns using real-time imaging based single cell analysis.Moreover,polyplexes were stable in the cytosol,entered the nucleus during mitosis and followed by dissociation and transcription,which could cause cells to sustainedly start expression.Compared with viral vectors,the primary cause for the low transfection mediated by cationic polymers is their inefficient dissociation and thus slow DNA release.Cationic polymers play mixed roles in gene delivery.While they are essential to pack large DNA coils into stable polymer/DNA complexes(polyplexes),protecting DNA from nuclease degradation and facilitating cellular uptake,their strong binding makes polyplexes hardly dissociate and DNA inefficiently transcribed.Cationic polymers even can interfere with transcription process.Therefore,in the fourth chapter,we proposed that a cationic polymer capable of degrading to uncharged fragments once inside cells would not only quickly release the DNA but also not interfere with the gene transcription process,resulting in efficient gene expression.We synthesized phenylboronic-containing polythioethers via "thiol-bromo" click reaction followed by sulfonization with trifluoromethane sulfonate methylate.By tailoring the monomer structures and feeding ratios,seven polysulfoniums with different molecular weights,hydrophobicity and ROS sensitivity were prepared and well characterized for the subsequent gene transfection.In the fifth chapter,we used the polysulfoniums for tumor gene delivery and evaluated the ROS-dependent transfection mechanisms.The polysulfoniums could condense DNA into uniform nano-sized polyplexes,which could be quickly internalized and efficiently escape from endo/lysosomes.In cancer cells,the oxidation of the boronic acid/ester by the elevated ROS levels triggered polysulfonium to break down into neutral thioether fragments,efficently releasing DNA for gene expression.The structure-transfection relationship was discussed,and the best performing polysulfonium 6CBE12k showed transfection efficiency 2-3 orders of magnitudes higher than bPEI25k.In vivo,6CBE12k polyplexes efficiently delivered the suicide gene pTRAIL to intraperitoneal tumors eliciting excellent anticancer activity. |
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