Oligonucleotide drug delivery, cellular response, and RNA sensing by biofunctional nanomaterials = 생기능성 나노물질에 의한 올리고핵산 약물전달, 세포 반응, RNA 감지에 관한 연구

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Gene therapy may be therapeutically useful in relieving symptoms and treating any kind of diseases which hybridize with their complementary target site in mRNA, blocking translation to protein expression of patho-physiologic genes. These strategies include DNAzymes, siRNA, antisense oligonucleotides (ASO), ribozymes, and aptamers. Among these molecules, deoxyribozymes (DNAzyme, Dz) have been paid attention as therapeutics both in cell-based assays and in preclinical models of diseases including cancer and viral infectious diseases. The “10-23” RNA cleaving DNAzyme has been shown to cleave any purine-pyrimidine junction under simulated physiological conditions, therefore efficiently inhibiting expression of target proteins in vitro and in vivo studies. These molecules ideally combine the catalytic activity of ribozymes with the stability of oligodeoxynucleotides, are easy to synthesize and less sensitive to chemical and enzymatic degradation than RNA-based reagents. Factors to influence the eventual therapeutic use of DNAzymes include its efficient cellular uptake, subcellular localization, and stability. A particularly important challenge to achieve the successful down-regulation of gene expression is to deliver DNAzymes efficiently to its intended site of action. Since various nanomaterials have unique, useful chemical, physical, and mechanical properties, they can be used for a wide variety of applications including nano-based biosensors, drug delivery devices, diagnostic tools, and means for tissue engineering and fundamental cell biology studies, etc. In chapter 2, we describe synthesis and characterization of a multifunctional magnetic nanoparticle (MION) for both noninvasive in vivo imaging and delivery of DNAzyme to target organ for hepatitis C treatment. Hepatitis C is one of the infectious diseases in the liver caused by the hepatitis C virus (HCV), a small-sized, enveloped, positive sense single strand RNA virus. The multifunctional nanoparticles consist of magnetic nanoparticles labeled with near-infrared fluorescent dye and conjugated to a synthetic DNAzyme targeting a gene of interest. In addition, these nanoparticles are tailored with cell-penetrating peptides (CPPs) helping membrane translocation process. We demonstrated the silencing effect of a gene of interest by Dz-loaded multifunctional nanoparticles in cultured human liver cells (Huh-7). For in vivo study in mice, we performed the alkaline phosphatase activity assay using sera to measure the efficiency of DNA transfection, and found that the hydrodynamic delivery of the reporter plasmid elicited and was maintained over-expression in mice for some periods. The delivery of the nanoparticles would be monitored in dual fashions in vitro and in vivo. We believe that our Dz-conjugated nanoparticles will be one of the widely applicable therapeutic options for the efficient HCV treatment in near future. Carbon based nanomaterials have shown much interest due to their unique structural and electrical properties such as mechanical strength, flexibility, electrical transport capability, young’s modulus, lightness and chemical inertness. Especially, graphene has the infinite possibilities to serve as novel nanoscale building blocks to create distinctive macroscopic materials. Due to their outstanding thermal and mechanical properties and high electrical conductivity, graphene sheets have been considered as a promising candidate for nanoelectronic devices, quantum computer, transparent electrode, and nanocomposite materials. Conductive substrate show great potential in biological applications such as tissue engineering, implants, drug delivery carriers, biochips for diagnostics and nano-devices for biological study. Substrates for immobilizing cells and tissues are valuable in use of biological and medical field study. The adhesion and spreading of mammalian cells is mediated by the binding of cell-surface integrin receptors to peptide ligands from the extracellular matrix (ECM) and the clustering of these receptors into focal adhesion complexes. Integrins play a critical role in the formation of focal adhesions, which attach cells to the extracellular matrix. It has been reported that the interaction between cells and ECM depends on the multiple substrate characters such as chemical composition, geometry, and topological aspects, ligand organization, and substrate stiffness. In addition, these factors of engineered substrates based on nanomaterials can affect and even lead to various cellular responses and cell physiology. Little is to investigate their properties that make the influence of carbon based nanomaterials including graphene sheet on living system. While the advancements in technology may be considerable, there is also concern about unintended effects of exposure to nanomaterials. In chapter 3, the chemically modified graphene oxides were immobilized on the glass substrates and used as a substrate for mammalian cells as a model of biological system to examine their influence on cell adhesion, spreading pattern and proliferation by various assays. We believe our result could serve as a fundamental standard for biological investigation of chemically prepared graphene-based nanomaterials. As biomolecules inside cells including proteins, nucleic acids, and small molecules show a variety of expression level and pattern, localization or distribution, they are considered as the critical parameters that reflect the state of organism including cellular behaviors, function, proliferation, development, physiological and pathological states. In that point of view, the build-up of the biomolecule detection method is one of the important issues in the biomedical field for the treatment process of all sorts of diseases. MicroRNAs (miRNAs) are a class of small-sizes (10~25nt) and non-coding RNA molecules that play an important regulatory role in the expression of diverse genes. Interestingly, miRNAs as the attractive biomolecule have been paid great attentions in a wide range of biological processes like development and metabolism and pathological progresses of disease/ disorders. Here, we fabricated the microRNA analytical platform for rapid, simple, and sensitive detection using nano-sized graphene oxide and detection probe PNA (peptide nucleic acid) for this study. In chater 4, we evaluated that 1) the probe-nanographene complex can work as microRNA sensing platform with efficient fluorescence quenching and recovery ability, 2) nanographene oxide sheets can serve as a delivery carrier of detection probe into live cells for real-time monitoring and quantitative analysis of microRNA and 3) the nanogrpahene oxide sheets provide the stable loading platform in the complex biological solutions and samples.
Advisors
Lee, Younghoonresearcher이영훈researcher
Description
한국과학기술원 :화학과,
Publisher
한국과학기술원
Issue Date
2012
Identifier
325007
Language
eng
Description

학위논문(박사) - 한국과학기술원 : 화학과, 2012.8,[xi, 122 p. :]

Keywords

Nanomaterials▼aoligonucleotides▼acellular response▼aMicroRNA; 나노물질▼a올리고핵산▼a세포 반응▼a마이크로 알엔에이

URI
http://hdl.handle.net/10203/265477
Link
http://library.kaist.ac.kr/search/detail/view.do?bibCtrlNo=848964&flag=dissertation
Appears in Collection
CH-Theses_Ph.D.(박사논문)
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