Prolonged in vivo gene silencing using multilayered nanoparticles as a sirna delivery system

Currently, there are various diseases without effective mode of cure. Many interventions are hampered by undesirable proteins produced over time. One example is the accumulation of excessive collagen protein at surgical site in post glaucoma surgery patients, rendering surgery failure. Excessive collagen accumulation results in scarring.Current anti-proliferative agents used such as 5 Fluorouracil and Mitomycin C lack specificity, indiscriminately affecting tissues adversely when applied topically or subconjunctivally injected, causing complications. Thus, there is a need for an effective anti-scarring agent with localized, specific action that also gives a prolonged effect over time. siRNA has been explored to prevent fibrosis but its use was limited due to the lack of stable and sustained delivery systems. This work reports on the successful development of a new nanocarrier system for the management of fibrosis. The new carrier has a hydroxy apatite core, with alternating layers of siRNA and a cationic peptide. While the siRNA silences a fibroblast gene responsible for producing a key mediator of fibrosis called SPARC, the nanocarrier is formulated to protect the siRNA, improve cell uptake and ensure specific prolonged silencing of genes for 2 weeks. The Layer by Layer nanoparticle of HA|ARG|siRNA|ARG|siRNA|ARG configuration was 242 nm in hydrodynamic diameter with 49.3 mV surface charge. The nanoparticle has a positively charged outermost layer to protect the siRNA and to bind electrostatically to cellular membranes when added to cells, facilitating cell uptake. FESEM and TEM imaging showed that the NPs were spherical, with smooth surfaces, smaller than 200 nm in diameter and uniform in size. The designed nanoparticles (at various dosages) did not cause any toxic effects on MCF cells over 2 weeks and in vitro studies showed sustained SPARC knockdown without affecting cell growth and maintained siRNA presence in cells for 2 weeks with a single dose treatment. In addition, in vivo studies in the mouse model of conjunctival scarring showed that a single dose injection into the conjunctiva facilitated SPARC knockdown, leading to prolonged efficacy of fibrosis suppression. Moreover, the nanoparticle treatment did not cause tissue apoptosis or foreign body response. Mechanism studies were conducted to investigate the breakdown behaviour of the particles, focusing on the release patterns of siRNA. This is the first demonstration of a stabilized siRNA delivery system showing sustained action against fibrosis over 2 weeks with a single injection.Doctor of Philosoph