Engineering glycogen-siRNA constructs with bioactive properties
AffiliationChemical and Biomolecular Engineering
Document TypePhD thesis
Access StatusThis item is embargoed and will be available on 2021-09-23.
© 2019 Marcin Wojnilowicz
RNA therapeutics, such as small interfering RNA (siRNA), have great potential for the treatment of inherited and acquired diseases that are not curable with conventional methods. The delivery of new genetic material into cells provides an opportunity to alter the expression of malfunctioning genes. However, siRNA is a hydrophilic and negatively charged molecule, which cannot easily cross biological membranes and is susceptible to degradation by nucleases present in biological fluids. Therefore, siRNA therapeutics require carriers that can effectively deliver their cargo into target cells. Early formulations for siRNA delivery involved systems based on viral vectors, lipid-based nanoparticles and cationic polymers. However, these formulations often displayed high toxicity, immunogenicity, instability in biological media, inability to penetrate tissue, and/or rapid clearance from the blood stream. Fine control over carrier size and surface properties, use of simplified and reproducible synthesis approaches, and deeper understanding of the interactions between siRNA-nanoconstructs in extra- and intracellular environment can potentially improve the engineering of new carriers. In this thesis, influence of the structural properties of soft glycogen nanoparticles on the formation of siRNA constructs and their delivery in a complex biological environment were investigated. Glycogen is a hyper-branched glucose bio-polymer of nanometer size that may be isolated from various animal tissues or plants. It is composed of repeating units of glucose connected by linear α-D-(1−4) glycosidic linkages with α-D-(1-6) branching. In this work, the properties of soft glycogen nanoparticles were tailored for the engineering of glycogen-siRNA constructs. These constructs were carefully designed to efficiently penetrate 3D multicellular tumour spheroids and exert a significant gene silencing effect. Obtained results suggest that 20 nm glycogen nanoparticles are optimal for complexation and efficient delivery of siRNA. The chemical composition, surface charge, and size of glycogen-siRNA constructs were finely controlled to minimize interactions with serum proteins which influence the stability and integrity of the glycogen-siRNA constructs. pH-sensitive moieties were introduced within the construct to enhance early endosomal escape. Using single molecule super-resolution microscopy, we demonstrate that the architecture of glycogen-siRNA constructs and the rigidity of the cationic polymer chains are crucial parameters that control the mechanism of endosomal escape driven by the proton sponge effect. The interactions of glycogen-siRNA constructs with immune cells were also investigated, suggesting that glycogen-siRNA constructs may be cleared from the blood stream by mononuclear phagocytic system, but can still successfully deliver the therapeutic cargo.
Keywordsglycogen particles; nanoparticles; gene delivery; RNA interference; endosomal escape; stochastic optical reconstruction microscopy; peripheral blood mononuclear cells; biological barriers
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