Multiple Acting Stable Sirna Nanomedicine Mediates Glioma Therapy

Malignant brain glioma is renowned as one of the most difficult types of tumor for treatment.This is due to the fact of their location of occurrence and thusly not only complete surgical removal is restrained,but also easily compressed for the nerve,therefore threatening normal human body function.By far,there are yet no effective clinical treatment plan for malignant brain glioma.A variety of treatment for glioma treatment are readily available,and nucleic acid drugs such as small interfering RNA(siRNA)are in the research hotspot now,playing an important role in the treatment of glioma.Especially,given the rapid development of nanotechnology greatly improved the siRNA delivery efficiency,clinical transformation of siRNA drugs were significantly accelerated.However,many challenges remain in the delivery process,such as blood stability,blood brain barrier(BBB),active tumor enrichment and in situ efficient release.Thusly,this project constructed a triple force-stabilized(charge,hydrogen bond,hydrophobic)siRNA nanomedicine(3I-NM@siRNA),which has the potential to treat glioblastoma through modification of brain-targeted ligand Angiopep-2(Ang).Electrophoresis experiments and dynamic laser light scattering results showed that 3I-NM@siRNA had better siRNA encapsulation and compression ability in forming smaller nanoparticles(38 nm),compared with the traditional single force(charge)stable siRNA nanomedicine(1I-NM@siRNA).The results of physiological stability experiment demonstrated that 3I-NM@siRNA had better stability under the competition of the negative-charged heparin sodium,and its blood circulation time(38 minutes half-life time)was also significantly improved compared with that of 1I-NM@siRNA(8 minutes half-life time).At the same time,due to the hydrophobic stable molecules used in this experiment have reactive oxygen species(ROS)response ability,3I-NM@siRNA can release the loaded siRNAs in simulated tumor microenvironment rapidly and effectively,achieving its optimized therapeutic effect.Experiments at cellular level results illustrated that,the Angiopep-2 polypeptide ligand modification enabled 3I-NM@siRNA with targeting ability for human glioma cell U87-MG.Meanwhile,flowcytometry quantitative analysis exhibited that the targeting efficiency of Ang-3I-NM@siRNA showed 3.5-fold changes of the non-targeting ones.At the same time,luciferase RNA interference cell experiment further confirmed the targeting ability of Ang-3I-NM@siRNA.The gene silencing efficiency of Ang-3I-NM@siRNA reached 80%,which showed significant increase than that of non-targeting nanoparticles(3I-NM@siRNA),under the same conditions(i.e.50%).Furthermore,MTT results showed that Ang-3I-NM@siRNA nanoparticles have ideal biocompatibility with minimum toxicity.To verify the therapeutic effect of Ang-3I-NM@siRNA,RNAi experiments targeting polo-like kinase-1(PLK1),qRT-PCR and Western Blot were performed.Excellent siRNA treatment effect were demonstrated by the fact that the gene silencing efficiency of Ang-3I-NM@siRNA achieved 80% and 63% at mRNA and protein level with 200 nM siRNA,respectively.In order to further verify the targeting and therapeutic effect of Ang-3I-NM@siRNA,an in situ human glioma mouse model was constructed.In the in vivo biological distribution study of siRNA nanomedicines,Ang-3I-NM@siRNA showed better targeting capacity for glioma,whose amount of accumulation at the tumor site was twice of that of 3I-NM@siRNA.When glioma-bearing mice were treated with siPLK1 and siVEGFR2(VEGFR2: vascular endothelial growth factor receptor),the results showed Ang-3I-NM@si(PLK1+VEGFR2)-treated mice had the slowest tumor growth,the least weight lost and the longest survival time(52 days).Histological H&E staining and TUNEL assay of tumor showed that Ang-3I-NM@si(PLK1+VEGFR2)group had the best proliferation inhibitory and elimination effect on glioma cells.When blood vessels were labeled with CD31(platelet-endothelial cell adhesion molecule),their immunofluorescence results demonstrated that the number of nascent blood vessels in tumor treated with Ang-3I-NM@siVEGFR2 and Ang-3I-NM@si(PLK1+VEGFR2)was significantly reduced.Moreover,siPLK1 and siVEGFR2 demonstrated an additive treatment effect of brain glioma.In conclusion,this research successfully constructed a triple force-stabilized brain-targeting siRNA nanomedicine which has good blood stability,high enrichment capacity in brain tumors and effective on-site siRNA release.Experiments at cellular and animal level have confirmed that therapeutic siRNAs-carrying nanomedicines have optimized gene silencing and tumor growth inhibitory effect.This smart siRNA nanomedicine has potential for clinical treatment of human brain glioma.