Self-assembled DNA Dendrimer Nanoparticle For Efficient Delivery Of Immunostimulatory CpG Motifs

The immunostimulatory cytosine-guanine dinucleotide sequence(CpG sequence)is an important tumor immunotherapy drug.However,naked CpG sequences show poor efficiency of cell internalization are prone to being degraded by nucleases.DNA dendrimers have received a great deal of attention as novel drug delivery systems recent years,owing to attractive properties such as mechanical stability,customized sizes and no detectable cytotoxicity.In this study,we use self-assembled DNA dendrimers as efficicent delivery vehicles for CpG sequences to enhance cell internalization and achieve the best immunostimulatory effect.1.DNA dendrimer deliver immunostimulatory CpGWe fabricated CpG loaded DNA dendrimer,then use MTT cell viability assay to verify the low cytotoxicity of DNA dendrimer.We analysed the cellular uptake of CpG loaded DNA dendrimer by confocal fluorescence microscopy,and CpG loaded DNA dendrimer showed higher cellular uptake efficiency than free CpG.The release of tumor necrosis factor(TNF-?)was detected by enzyme-linked immunosorbent assay(ELISA),and the CpG loaded DNA dendrimer could induce the higher release of cytokines.2.Functional DNA dendrimer used in CpG delivery and immunotherapyWe fabricated CpG and TAT peptide functionalized DNA dendrimerto deliver CpG-containing hairpin-loops.We analysed the cellular uptake and location of CpG loaded functional DNA dendrimer by flow cytometry and confocal fluorescence microscopy,and TAT peptide functionalized DNA dendrimer was efficient in cellular uptake and endosome location.TAT decorated DNA dendrimer induced the highest release of TNF-? and interleukin-6(IL-6)and DNA dendrimers decorated with CpG-containing hairpin-loops triggered stronger immune response characterized by pro-inflammatory cytokines production,in contrast to DNA dendrimers loading with CpG in linear-structure.CpG loaded DNA dendrimer showed higher cellular uptake efficiency than free CpG.The release of tumor necrosis factor(TNF-?)was detected by enzyme-linked immunosorbent assay(ELISA),and the CpG loaded DNA dendrimer could induce the higher release of cytokines.The formation of functional DNA nanostructure was confirmed by gel electrophoresis,dynamic light scattering and atomic force microscopy.Cytotoxicity assay showed that this nanoparticle did not affect the viability of macrophages.Confocal microscopic images and flow cytometry results showed that DNA dendrimer significantly enhance the internalization quantity of CpG ODNs into cells compared to short DNA single strands.The results from pro-inflammatory cytokines showed that compared to linear-structure CpG both single and double strands,TAT-mediated hairpin-loop containing CpG motifs cause high immunostimulatory activity.These results illustrate that DNA dendrimers can effectively increase the uptake of CpG cells.In additon,through chemical attachment of cell-penetrating peptides(TAT peptides)on DNA dendrimers,multifunctional drug-delivery systems can further enhance the biomembrane-crossing rates and the internaLization quantity of drugs into cells and functional DNA dendrimer are more effective in stimulating the release of cytokines,thereby activating the immune response.DNA dendrimers decorated with CpG-containing hairpin-loops triggered stronger immune response characterized by pro-inflammatory cytokines production,in contrast to DNA dendrimers loading with CpG in linear-structure.Our results proved that TAT modified DNA dendrimers are promising drug delivery tools in immunotherapy.