Author

Chao Liang

Year of Award

2016

Degree Type

Thesis

Degree Name

Doctor of Philosophy (PhD)

Department

School of Chinese Medicine.Teaching and Research Division.

Principal Supervisor

Zhang, Ge

Keywords

Bones;Diseases;Disorders;Gene therapy;Metabolism;Osteoclasts

Language

English

Abstract

Osteoporosis remain major clinical challenges. RNA interference (RNAi) provides a promising approach for promoting osteoblastic bone formation to settle the challenges. However, the major bottleneck for translating RNAi with efficacy and safety to clinical bone anabolic strategy is lack of osteoblast-specific delivery systems for osteogenic siRNAs. Previously, we developed a targeting system involving DOTAP-based cationic liposomes attached to oligopeptides (AspSerSer)6, (also known as (DSS)6), which had good affinity for bone formation surface. Using this system, osteogenic Pleckstrin Homology Domain Containing, Family O Member 1 (Plekho1) siRNA could be specifically delivered to bone formation surface at tissue level and promoted bone formation in osteopenic rodents. However, concerns still exist regarding indirect osteoblast-specific delivery, detrimental retention in hepatocytes, mononuclear phagocyte system (MPS)-induced dose reduction and inefficient nanoparticle extravasation. Aptamers, selected by cell-based Systematic evolution of ligands by exponential enrichment (cell-SELEX), are single-stranded DNA (ssDNA) or RNA which binds to target cells specifically by distinct tertiary structures. By performing positive selection with osteoblasts and negative selection with hepatocytes and peripheral blood mononuclear cells (PBMCs), we aimed to screen an aptamer that could achieve direct osteoblast-specific delivery and minimal hepatocyte and PBMCs accumulation of Plekho1 siRNAs. In addition, lipid nanoparticles (LNPs) have been widely used as nanomaterials encapsulating siRNA due to their small particle size below 90 nm. Polyethylene glycol¡(PEG) as the mostly used hydrophilic polymer, could efficiently prevent LNPs from MPS uptake. So, LNPs with PEG shielding could serve as siRNA carriers to realize efficient extravasation from fenestrated capillaries to osteoblasts and help reduce MPS uptake of the siRNAs. Recently, we screened an aptamer (CH6) by cell-SELEX specifically targeting both rat and human osteoblasts and developed the aptamer-functionalized LNPs encapsulating osteogenic Plekho1 siRNA, i.e., CH6-LNPs-siRNA. Our results demonstrated that CH6-LNPs-siRNA had an average particle size below 90 nm and no significant cytotoxicity in vitro. CH6 aptamer facilitated osteoblast-selective uptake of Plekho1 siRNA and gene silencing in vitro. In this study, we further found that CH6 aptamer facilitated the bone-specific distribution of siRNA by biophotonic imaging and quantitative analysis. Immunohistochemistry results showed that CH6 achieved in vivo osteoblast-specific delivery of Plekho1 siRNA. Dose-response experiment indicated that CH6-LNPs-siRNA achieved almost 80% gene knockdown at the siRNA dose of 1.0 mg/kg and maintained 12 days for over 50% gene silencing. microCT, bone histomorphometry and mechanical testing confirmed that CH6 facilitated bone formation, leading to improved bone micro-architecture, increased bone mass and enhanced mechanical properties in osteoporotic rodents. Furthermore, CH6-LNPs-siRNA achieved better bone anabolic action when compared to the previously developed (AspSerSer)6-liposome-siRNA. There was no obvious toxicity in rats injected with CH6-LNPs-siRNA. All these results indicated that osteoblast-specific aptamer-functionalized LNPs could act as a novel RNAi-based bone anabolic strategy and advance selectivity of targeted delivery for osteogenic siRNAs from tissue level toward cellular level. In addition, the generation of ssDNA from double-stranded PCR products is an essential step in selection of aptamers in SELEX. We found that the size separation derived from unequal primers with chemical modification could be a satisfactory alternative to the classic magnetic separation.

Comments

Principal supervisor: Dr. Zhang Ge.;Thesis submitted to the School of Chinese Medicine Teaching and Research Division.;Thesis (Ph.D.)--Hong Kong Baptist University, 2016.;

Bibliography

Includes bibliographical references (pages 105-117)

Available for download on Tuesday, February 27, 2018



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