• Development of Spray Dried Microparticulate Delivery Systems for Vaccines and Oligonucleotides

      Ubale, Ruhi V; College of Pharmacy
      In recent years, research related to the development of biotherapeutics has gained tremendous interest in industry as well as academia. There are a number of biologics available in the market and many more currently being evaluated in clinical trials. Their development has gained momentum due to the range of advantages they offer over traditional small molecule drugs. However, there still remain challenges with their delivery mainly due to the poor stability of these molecules in harsh physiological conditions. A number of strategies are being devised for the delivery of biomolecules by non-invasive routes to make delivery easier and more patient compliant. Microparticles are one of the options being explored as they offer protection, sustained release and enhanced bioactivity to the biomolecule. In this project, we have studied the use of microparticles for the development of formulations for a meningococcal polysaccharide vaccine and an antisense oligonucleotide to NF-kB. Meningitis occurs majorly in children and is caused by Neisseria meninigitidis. Infection by this bacterium spreads rapidly through the body resulting in loss of limbs, hearing and can also be fatal. Hence, vaccination is considered as the only means prevention for meninigitis. Marketed vaccines use protein conjugated bacterial capsular polysaccharides as the antigen. Polysacchatides are considered inherently weak antigens with a T- independent immune response. With the development of a microparticulate vaccine using a protien matrix, we expected to stimulate the T-dependent immune pathway for these antigens avoiding the use of a conjugated Diphtheria toxoid. We have studied the enhancement of innate immune responses by the microparticulate vaccine. The antigen used N. meningitidis polysaccharide A, aslo an adjuvant- kdtA(unglecosylated lipid A)- was studied for its response. The microparticulate vaccine was characterized for its physicochemical characteristics like size, charge and toxicity. Also it was studied for the innate immune responses the microparticles can elicit after incubation with murine and human macrophages. Release of innate immune markers like TNF-a, IL-1B, IL-8, nitric oxide and reactive oxygen species from macrophages was studied and the microparticulate vaccine was observed to elicit a desirable innate immune response. Antigen presentation by macropaphages stimulated with the microparticulate vaccine was also studied by visualizing autophagy. Lung inflammation occurs doe to an infection or a damaging agent that irritates the lung lining or pleura. NF-kB is a nuclear transcription factor activated by stimuli like endotoxin and bacteria that initiates the synthesis of pro inflammatory cytokines such as TNF-a, IL-1, IL-6, etc. Antisense are single stranded RNA complementary to a chosen sequence that offer great potential to inhibit the synthesis of individual proteins by interfering with protein translation. As intracellular penetration of antisence compounds has proven to be a limiting factor in their effectiveness, we have proposed to develop a microparticulate formulation for pulmonary delivery of antisense to NF-kB and evaluate their efficacy. The microparticulate formulation of antisense to NF-kB was characterized for its physiochemical characteristics like size, charge, antisense content and release. Since enhancing uptake into cells was the crux of the study, we also visualized and quantified uptake of microparticles by microphages. We looked at the period of lung resistance and the biodistribution of the microparticles using a near infrared bioimager. Serum levels of proinflammatory cytokines were studied after induction of lung inflammation in a rat model. Animals that were delivered antisense microparticles to the lung showed reduced levels of TNF-a and IL-1b.