RESUMEN
siRNA is currently the most widely studied form of RNAi, and it has promising therapeutic potential not just in cancer but also in other diseases such as autoimmune and infectious diseases. However, efficient delivery of siRNA to target cells is being limited by lack of an effective delivery system that ensures efficient transfection into cells while protecting the encapsulated siRNA from nuclease. We hypothesized that a hybrid nanoparticle system composed of human IgG and poloxamer-188, a stealth polymer, will efficiently deliver mutated KRAS siRNA to A549 cells, leading to an efficient knockdown of mutated siRNA while protecting the siRNA from serum nuclease. We also hypothesized that the nanoparticles will not elicit an immunostimulatory effect in murine macrophages and also avoid clearance by macrophages. These nanoparticles were found to efficiently deliver siRNA to the cytoplasm and nuclease of A549 cells in a controlled and sustained manner while avoiding recycling by endosomes. An effective knockdown of mutated KRAS was achieved, which subsequently led to an increased sensitivity to erlotinib. These nanoparticles successfully avoided uptake by murine macrophages and reduced immune responses normally associated with siRNA/nanoparticle therapy. These results demonstrate that the novel hybrid nanoparticles could potentially serve as a platform for efficient delivery of siRNA to cells for stable gene knockdown.
Asunto(s)
Nanopartículas/química , Proteínas Proto-Oncogénicas/genética , ARN Interferente Pequeño/química , ARN Interferente Pequeño/genética , Transfección/métodos , Proteínas ras/genética , Línea Celular Tumoral , Técnicas de Silenciamiento del Gen , Humanos , Proteínas Proto-Oncogénicas p21(ras)RESUMEN
Therapeutic aerosol bioengineering (TAB) of Mycobacterium tuberculosis (MTb) therapies using inhalable microparticles offers a unique opportunity to target drugs to the site of infection in the alveolar macrophages, thereby increasing dosing in the lungs and limiting systemic exposure to often toxic drugs. Previous work by us used sophisticated, high content analysis to design the optimal poly(lactide-co-glycolic) acid (PLGA) microparticle for delivery of drugs to alveolar macrophages. Herein, we applied this technology to three different anti-MTb drugs. These formulations were then tested for encapsulation efficiency, drug-release, in vitro killing against MTb and aerosol performance. Methods for encapsulating each of the drugs in the PLGA microparticles were successfully developed and found to be capable of controlling the release of the drug for up to 4 days. The efficacy of each of the encapsulated anti-MTb drugs was maintained and in some cases enhanced post-encapsulation. A method of processing these drug-loaded microparticles for inhalation using standard dry powder inhaler devices was successfully developed that enabled a very high respirable dose of the drug to be delivered from a simple dry powder inhaler device. Overall, TAB offers unique opportunities to more effectively treat MTb with many potential clinical and economic benefits resulting.