| Ever since the first representation of RNAi phenomenon in 1998, its ride has been quite complicated. If handled carefully, RNAi could have the potential of transforming into a therapeutic which can apply in a wide range of diseases from viral to cancer. The use of a powerful therapeutic tool like siRNA therapeutics has faced many barriers, like: molecule stability, specific delivery to desired cells and tissues, triggering of innate immune response, and effects achieved off the desired site. Theoretically, siRNA can be used to silence any gene, therefore is a potential therapeutic to many genetic diseases, and not only. However, given the enormous genetic variation between species RNAi becomes a challenge. Sepsis syndrome is one of those diseases that quickly progresses over time. There is a demanding need in finding a therapy for advanced sepsis syndrome, a fast-running progressive disease. The main mediators of inflammation in general and sepsis in particular like, TNF-alpha and IL1 play an important role in triggering a spillover of a flood of cytokines leading to sepsis condition. Anti-TNF-alpha therapies have been applied in inflammatory diseases and in sepsis in a long time, but despite the promising results of the in vivo work the translation to humans has been difficult. From its role prospects, TNF-alpha gene is a tricky target to modulate, and siRNA therapy offers transitory effect instead of an enduring effect over this target. We developed a nanosized delivery system, made of modified hyaluronic acid polymer encapsulating cholesterol modified siRNA in its core, to enable the siRNA supply in the macrophages` cytoplasm. The particle we developed was characterized and a size of nanometer range was determined, which intact the siRNA through charge and hydrophobic interaction. The main goal of this thesis work was to develop a therapy for sepsis utilizing RNAi to target TNF-alpha, based on biomarkers identification to follow on the disease progression. One important goal achieved was the creation of a multimodal delivery system, to overcome the long known obstacle of siRNA delivery in cells cytoplasm. More specifically, targeting macrophage cells is a complicated task. Despite their ability to take up quickly, they also possess the high capability of degrading the engulfed materials. Nanotechnology principles are very helpful in generating delivery vehicles with unique physicochemical and biological properties. As a nanosized system, the formulation offered by this thesis work represents a combinatorial approach that contains poly(ethylene imine) (PEI) modified hyaluronic acid polymer in combination with lipid modified polymer. A poly(ethylene glycol) (PEG)-modified polymer is used as a component of the formulation to provide more stability given the negative charge of the siRNA. On one hand, the positively charged PEI plays an important role in encapsulating the siRNA through charge interaction, while on the other hand it contributes a proton sponge effect within the cells that facilitates the siRNA delivery into cytoplasm after escaping the late endosome. The chemical modification of the siRNA, by addition of a cholesterol molecule, and addition of lipid modified hyaluronic acid polymer, lead to improvement of encapsulation efficacy, while enhancing the TNF-alpha silencing efficacy in peritoneal macrophages. The silencing efficacy was supported by the biodistribution data in vivo, which showed efficacious siRNA uptake preferably in peritoneal macrophages, as compared to other organs tissues in mice. Septic mice treated with anti- TNF-alpha siRNA nanoparticle showed a significantly improved survival rate, as compared to septic mice treated with scramble siRNA nanoparticle, in a LPS developed model of sepsis. Thus, the novel multifunctional nanoparticle encapsulating siRNA, offers an alternate, safe, and effective siRNA delivery system to treat sepsis, and in more general terms, the inflammatory diseases. |
