Nucleic Acid Drug And Drug Delivery Vector Based On RNA Interference Technology For Cancer Therapy

RNA interference (RNAi) is a common biological phenomenon. In the research and development of small nucleic acid drugs based on RNAi, the studies should focus on discovering new siRNAs and miRNAs and fully exploring the function and mechanism of siRNAs and miRNAs for clinical applications. Furthermore, due to its fast degradation in the physiological environment, poor cellular uptake, inefficient translocation into the cytoplasm, and lack of targeting ability, delivery of synthetic small RNA remains the major obstacle to its therapeutic application. Development of effective delivery vectors is therefore essential for RNAi-based therapy. This dissertation can be categorized into three main parts as described below:1. Breast cancer metastasis is the main cause of death in breast cancer patients. MicroRNAs (miRNA) have been documented playing a critical role in cancer development and progression. In this part, we have found that miR-190was frequently downregulated in highly metastasic human breast cancer. Further studies have shown that overexpression of miR-190suppress breast cancer cell migration and invasion in vitro and tumor metastasis in vivo. Transcription factor4(TCF4) and rho-associated, coiled-coil containing protein kinase1(ROCK1) are identified as direct and functional targets of miR-190. In addition, overexpression of miR-190in MDA-MB-231cells can reduce the protein levels of TCF4or ROCK1. Luciferase assays confirm that miR-190could directly bind to the3’untranslated region of TCF4or ROCK1. Moreover, knockdown of TCF4or ROCK1significantly inhibits breast cancer cell migration and invasion resembling that of miR-190overexpression. The data from the current study suggest that miR-190acts as a novel metastasis suppressor in breast cancer and that downregulated miR-190contributes to distant metastasis.2. An efficient and safe delivery system for small interfering RNA (siRNA) is required for clinical application of RNA interfering therapeutics. Polyethyleneimine (PEI)-capped gold nanoparticles (AuNPs) have been successfully manufactured using PEI as the reductant and stabilizer, which bind siRNA at an appropriate weight ratio by electrostatic interaction and result in well-dispersed nanoparticles with uniform structure and narrow size distribution. With siRNA binding, PEI-capped AuNPs induce more significant and enhanced reduction in targeted green fluorescent protein expression in MDA-MB-435s cells, though more internalized PEI/siRNA complexes in cells are evidenced by confocal laser scanning microscopy observation and fluorescence-activated cell sorting analyses. PEI-capped AuNPs/siRNA targeting endogenous cell-cycle kinase, an oncogene polo-like kinase1(PLK1), display significant gene expression knockdown and induce enhanced cell apoptosis, whereas it is not obvious when the cells are treated with PLK1siRNA using PEI as the carrier. Without exhibiting cellular toxicity, PEI-capped AuNPs appear to be suitable as a potential carrier for intracellular siRNA delivery.3. Novel diblock copolymers of poly(ε-caprolactone) and polyphosphoester bearing functional hydroxyl pendant groups, denoted as PCL-b-PHEP, have been synthesized through ring-opening polymerization of functional ized cyclic phosphoester monomer using hydroxyl end-capped poly(ε-caprolactone) and Sn(Oct)2as macroinitiator and catalyst, respectively. The chemical structure has been proven by1H,13C, and31P NMR, gel permeation chromatography (GPC), and Fourier transform infrared spectroscopy (FT-IR) analyses. These amphiphilic functionalized block copolymers can self-assemble into micellar or vesicular aggregates in aqueous solution, depending on the composition, which is demonstrated by transmission electron microscopy and confocal laser scanning microscope observations. The critical aggregation concentrations (CAC) of PCL-b-PHEP are also dependent on the composition, measured by the fluorescence probe technique. MTT assay for cytotoxicity of PCL-b-PHEP suggests these polymeric nanoparticles are biocompatible. Combining the advantages of poly(ε-caprolactone) and polyphosphoester with functional hydroxyl pendant groups for further biological modification, such amphiphilic block copolymers can potentially provide novel opportunities for design of drug delivery system and therapeutic application.