Nanomedicine Of Hyaluronic Acid Coordinated SiRNA For The Efficient Treatment Of Glioblastoma

RNA interference?RNAi?by using small interfering RNAs?siRNAs?to suppress the activity of specific gene is a current novel therapeutic strategy for cancer therapy.However,one of the most challenging aspect in clinical translation and practical application rests on targeted delivery of siRNAs.By far,the most renowned siRNA delivering system largely limited to cationic materials,for instance,polymers,lipids,and inorganic nanoparticles.While these strong cationic materials are capable of condensing anionic siRNAs into nanoparticles via electric interactions,as well as protecting siRNAs from RNase degradation during in vivo administration.Major downfall of these strong cationic carriers is the fact that they often induce severe cytotoxicity to cells and tissues.Therefore,the development of a weak cationic carrier for safe and efficient siRNA delivery is one of the most aspiring application prospects for cancer treatment.In this project,a less-cationic siRNA delivery carrier with desirable bioreducibility was designed.Hyaluronic acid?HA?was employed as backbone,linked with dipicolylamine?DPA?via redox sensitive disulfide?-SS-?linker.HA-SS-DPA conjugates are able to formulate siRNA nanocomplexes with excellent stability,by specifically chelate with siRNA in the presence of zinc ions.Once these nanocomplexes were taken up by tumor cells,the abundant intracellular reduction stimulus?i.e.glutathione;GSH?trigger the release of siRNAs and subsequent desired RNAi effect.In this research project,the biophysical characteristics,reduction responsiveness,endocytosis,pharmacokinetics and biocompatibility of this novel siRNA nanomedicine,as well as their in vitro and in vivo gene silencing efficiency and antitumor efficacy towards U87MG glioblastoma xenograft tumor models were extensively studied.First of all,HA-SS-DPA conjugates with different degrees of substitution were studied for their self-assembly with siRNAs in the forms of their biophysical characteristics.i)a serious of weight ratios?40:1,30:1,20:1 and 10:1?between HA-SS-DPA polymer and siRNAs were tested for self-assembly in the presence of Zn²⁺.Final product of HA-SS-DPA?Zn?/siRNA nanomedicine were found with an averaged size of 136 nm²33 nm measured with dynamic light scanning?DLS?;ii)Gel electrophoresis showed that after treated with 10 mM reduction agent DL-Dithiothreitol?DTT?,a complete release was observed for the coordinated siRNAs from HA-SS-DPA?Zn?/siRNA nanocomplexes,further proved the reduction responsivity of this nanocomplex;iii)A uniform spherical morphology and particle size at about 100 nm were observed for these nanocomplex under transmission electron microscope?TEM?.Secondly,the in vitro cytotoxicity,cellular uptake and gene knockdown effect of HA-SS-DPA?Zn?/siRNA nanocomplexes were investigated.i)A survival rate of 92.1⁹9.6%were achieved in the cytotoxicity experiments,confirmed the good biocompability of HA-SS-DPA?Zn?/siRNA nanocomplexes;ii)By analysing siRNA nanomedicine uptake by tumor cells using confocal microscopy and flow cytometry,this nanocomplex were found with enhanced uptake efficiency with increased DPA degree of graft;iii)A silencing efficiency of this particular encapsulated siRNA nanocomplex was found to achieve as high as48%in glioma cells?U87-Luc?.Last but not least,this project further investigated the pharmacokinetics,biodistribution,gene silencing effect and anti-tumor effect of HA-SS-DPA?Zn?/siRNA nanocomplex on the tumor-bearing model of xenograft glioblastoma.Pharmacokinetic results showed that the half-life of HA-SS-DPA?Zn?/siRNA was about 34.7 min,which was significantly higher than that of the free siRNAs?i.e.1.6 min?;ii)The qualitative and quantitative analysis of in vivo gene silencing results showed a 48%efficiency,identical to that of in vitro cell experiment;iii)Distribution of HA-SS-DPA?Zn?/siRNA nanocomplex were maily accumulated in the tumor site,in clear contrast to the naked siRNAs that were mostly distributed in kidney and liver;iv)Tumor inhibition growth assays were evaluated by targeting Polo-like kinase-1?PLK1?.The results showed that HA-SS-DPA?Zn?/siRNA could effectively restrain tumor growth;v)Lastly,histology analysis revealed that HA-SS-DPA?Zn?/siRNA caused widespread necrosis in tumor tissues,whereas little damage was found to the heart,liver,spleen,lung,and kidney.This further confirmed that HA-SS-DPA?Zn?/siRNA is equipped with specific tumor selectivity without toxic sideeffects.This study demonstrated that as a novel siRNA delivery complex,HA-SS-DPA?Zn?/siRNA nanocomplex shined with a set of key enhancement,including prolonged in vivo circulation half-life,enhanced cellular uptake,specific targeting,efficient siRNA release in lesions and achieved desiable treatment,thus present itself as a favorable tumor therapy with promising appilication prospects.