Well-defined Antisense Miktoarm Fullerenes For Enzyme-resistant Nanoflares And Carrier-free Gene Regulation

Oligonucleotides?ONs?are a promising class of agents in the diagnosis and treatment a wide variety of diseases including cancers,neuropathies,and metabolic disorders which are hard to cure.Difficult biopharmaceutical characteristics of unmodified ONs,such as rapid clearance by reticuloendothelial system,poor nuclease stability,low efficiency of cellular uptake,and the risk of activation of unwanted immune response,limit the translation of oligonucleotide drug to clinical practice.Although a series of synthetic transport carriers?cationic polymers,liposomes,nanoparticles,etc.?and chemical modified oligonucleotides have been developed to facilitate the delivery of ONs in body,a series of problems,such as insufficiency and high cytotoxicity,need to be solved.Therefore,there is still a great need for safe and effective ON delivery system that can be used systemically.In this thesis the research work mainly includes the following two sections:1.We designed and preparaed a class of well-defined poly?ethylene glycol?-oligonucleotide miktoarm star polymers(hereinafter referred to as C₆₀pacDNA),which were based on a 12 azido-fuctionalized fullerene C₆₀ core.The core is relatively compact but not overwhelmingly small?⁵ nm,assuming all bonds in the linkers are in a fully stretched conformation?,making it possible to fully derivatize the core and at the same time reach a high PEG density.DBCO-modified DNA and PEG chains were attached on the surface of C₆₀ core by a copper-free click reaction,respectively.One DNA strand can be attached by controlling the feeding ratio of DBCO-DNA and C₆₀ core.The resulted conjugates were purified by reverse-phase high-performance liquid chromatography?RP-HPLC?.Furthermore,the excess DBCO-PEGs?Mn=5,10 kDa,PDI<1.03?were fed to react with the remaining azide groups on the C₆₀-DNA conjugates completely.The resulted miktoarm star polymer C₆₀ pacDNAs containing 1 DNA strand and 11 PEG chains were purified by gel permeation chromatography?GPC?,and characterized by agarose gel,dynamic light scattering?DLS?,atomic force microscope?AFM?,fourier transform infrared spectroscopy?FT-IR?.2.An antisense DNA sequence against the human epidermal growth factor receptor 2?HER2?transcript is selected as a model strand.The biological characteristics of the synthesized star polymer C₆₀0 pacDNAs and the pacDNA nanoflares which were prepared by using C₆₀0 pacDNAs as building blocks,were investigated.First the ability of the C₆₀ pacDNAs to retard nuclease degradation were explored.C₆₀ pacDNA_10k bearing longer PEG chains and C₆₀ pacDNA_5k-m attaching the DNA via a midchain anchor exhibited extended 9.0×and 4.5×longer DNase I half-life compare to free DNA,respectively.And the C₆₀ pacDNA_10k and C₆₀pacDNA_5k-m effectively regulated cellular gene expression without the need for a cationic vector system,even better than the commercialized Lipofectamine^TM.These molecular entities consist of a single DNA strand,11 PEG chains and a fullerene C₆₀core with signature features associated with their polydisperse counterparts,such as selective binding to complementary DNA strands,increased nuclease resistance and increased cellular uptake.The C₆₀ pacDNA nanoflares exhibit a lower background than regular nanoflares,due to the improvement of nuclease stability.The successful synthesis and demonstration of these monodisperse structures are p oised to enable more detailed studies on their interactions with biosystems,and pave the way for potential translational studies.