Date

2018

Author

Sezlev Bilecen, Deniz

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Osteoporosis, the most common disease of bone, is a skeletal disorder associated with low bone mass, increase in bone fragility and in susceptibility to fractures. The high bone resorption rate is shown to be due to increased number and activity of the osteoclasts. Receptor Activator of Nuclear Factor kappa B (RANK)/ Receptor Activator of Nuclear Factor kappa B Ligand (RANKL) system plays a crucial role in osteoclast differentiation and bone remodeling. RANKL participates in differentiation and activation of osteoclasts by binding to its receptor RANK expressed on osteoclast progenitors and mature osteoclasts. The currently used drugs for osteoporosis, such as use of bisphosphonates, Denosumab and teriparatide, have low bioavailability, long-term safety concerns and cause gastric problems. This led to the need for delivering the drugs in carrier systems to increase their bioavailability and decrease their side effects. In recent years, however, the increased understanding of molecular background of bone pathology, the use of RNA interference (RNAi) gained interest in developing new treatment strategies for the disease. Small interfering RNA (siRNA) is a double stranded RNA (dsRNA) molecule which is used in this context to inhibit the translation of abnormal gene expression in cells. In this study, we developed a potentially intravenously injectable siRNA-delivery system that can target osteoclasts in bone tissue for the treatment of osteoporosis. Polyethyleneimine (PEI), a polycationic molecule, was used as a complex with RANK siRNA which was then loaded into poly(lactic acid-co-glycolic acid) (PLGA) nanocapsules. The loaded nanocapsules were coated with a genetically engineered osteoconductive polypeptide, Elastin like recombinamer (ELR) designed to attach especially to bone minerals, to specifically target the drug loaded nanocapsules to the bone tissue. The carrier system constructed was studied by Scanning Electron Microscopy (SEM), (Transmission Electron Microscopy) TEM and X-Ray Photoelectron Spectroscopy (XPS) to show the ELR coat on the capsules. Different nitrogen to phosphate ratios (N/P) (presenting PEI and siRNA, respectively) for the PEI:RANK siRNA complexes were studied to determine the least toxic and most effective complex which would lead to satisfactory RANK mRNA inhibition. As a result of that study, the N/P ratio of 20 was chosen to construct the complex to be loaded in the PLGA nanocapsules. The encapsulation efficiency of the PEI:RANK siRNA complex (N/P 20) into PLGA nanocapsules were 48% and its release kinetics followed the Higuchi kinetics for 15 days. The PEI:RANK siRNA loaded PLGA nanocapsules significantly inhibited RANK mRNA (53 %) in an osteoclast precursor cell line. The differentiation of the precursors into mature osteoclasts was also suppressed by the delivery system. Treatment of differentiating osteoclasts with the PEI:RANK siRNA loaded PLGA nanocapsules inhibited their osteoclastic activity. In conclusion, the delivery system designed has the potential to serve as an alternative treatment method for use in the treatment of osteoporosis.

Subject Keywords

Osteoporosis., Bones, Small interfering RNA.

URI

http://etd.lib.metu.edu.tr/upload/12622054/index.pdf
https://hdl.handle.net/11511/27261

Collections

Graduate School of Natural and Applied Sciences, Thesis