Targeted Microrna Delivery Based On Novel Nanotechnology And Its Application In Cancer Therapy

High incidences of esophageal squamous carcinoma （ESCC） and breast cancerhave been reported in China. Surgical therapy is mainly used for the treatment ofearlier stage cancers, while chemotherapy is usually used to treat late stage cancers.However, chemotherapy is challenged by high toxicity and drug resistance. Noveltherapeutic technology remains a great interest in tumor treatment domain.MicroRNAs （miRNA）, known as a family of small, highly conservedsingle-stranded noncoding RNA molecules, bind to the3’-untranslated region（3’-UTR） of mRNA through partial base-pairing and result in degradation andtranslation suppression of its target mRNA. The dysregulation of miRNA expressionhas been found to be closely associated with carcinogenesis and miRNAs function astumor suppressors or oncogenes during tumor initiation and progression. Interferingtechnology provides a great opportunity for tumor therapy. Nevertheless,miRNA-based therapy is challenged by delivery system. In this dissertation, miR-203and miR-34a, which are potent tumor suppressors in esophageal cancer and breastcancer, respectively, were loaded into nanosystems and delivered into tumor cells andtumor tissues. Moreover, folate acid（FA）was used to construct targeted nanosystem,whose tumor targeting ability and tumor suppressive activities were investigated invitro and in vivo.The major works of the dissertation include:A self-assembled approach by utilizing ND, PS and miR-203was used to producenanodrug miR-203/PS@NDs. ND surface was functionalized by acid treatment toenable the formation of numerous hydrogen bonds suiTab. for the attachment ofarginine-rich polycationic peptide-protamine, which have the ability to absorbmiR-203. The characteristics of miR-203/PS@NDs were examined by DLS（dynamic light scattering） assay and TEM （transmission electron microscope）,and the mass ratio of miR-203:PS@NDs was optimized by AGE （agarose gelelectrophoresis）. Besides, nanoparticle-encapsulated miR-203is sTab. in the presenceof serum and the release efficiency of miR-203increased in acid condition.In the second part of this dissertation, anti-tumor activity of miR-203/PS@NDwas examined by in vitro and in vivo models. Cytotoxicity and trancfectionexperiments showed that miR-203/PS@ND has a good biocompatibility and miR-203can be delivered into cells with a higher transfection efficiency than commercialtransfection agent PEI25K. Real-time PCR experiments demonstrated that miR-203suppressed cell proliferation and migration through targeting Ran and ΔNp63. In vivoexperiments showed that miR-203/PS@NDs inhibited tumor growth by either intratail vein or intratumoral administrations. However, intratumoral administration showed animproved tumor suppression than intratail vein.In the forth part of this dissertation, anti-tumor activities of nanodrugFA/PS/miR-34a/PS@NDs were measured by both in vitro and in vivo models. In vitroexperiments showed that miR-34a inhibited cell proliferation, migration and inducedcell apotosis through targeting Fra-1expression. In vivo experiments confirmed thatFA/PS/miR-34a/PS@NDs could inhibit tumor growth and enhanced cell apoptosis intumor issues by either intratumoral or intratail vein administrations. Moreover, thelocalization of nanoparticles was significantly observed in tumor tissues, suggesting atumor specific targeting ability of nanoparticles.In the present study, miR-203can be efficiently encapsulated bynanodiamond-based nanoparticles, which showed potent tumor suppressive activitiesagainst esophageal cancer. Otherwise, layer by layer nanoparticles were constructedthrough self-assembled approach by immobilizing FA on the surface of nanoparticles.Both in vitro and in vivo experiments showed that miR-34a-loaded nanoparticlessuppressed tumor growth through specifically targeting tumor cells.