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Synthesis of inorganic-biodegradable polymer composite microspheres for controlled delivery of a DNA prime-protein boost vaccine
thesisposted on 2017-01-09, 04:51 authored by Ho, Jenny
The demand for an efficient delivery system for plasmid DNA-based vaccines has increased vastly in response to the rapidly growing use of plasmid DNA (pDNA) as a non-viral vector for vaccination. However, several limitations such as ineffective cellular uptake and intracellular delivery, and degradation of pDNA need to be overcome. Intranasal vaccination has been an area of interest for the pharmaceutical industries in recent years to overcome the alarming pattern of unsafe injection practices, and the poor availability of injectable and orally administered vaccines. The advent of particulate delivery systems for the administration of pDNA through intranasal inhalation is relatively new. In this study, a novel and scalable technique has been developed to create an inorganic-biodegradable polymer composite system, which enables controlled delivery of a heterologous prime-boost vaccine via intranasal vaccination. This delivery system offers to increase the potency of pDNA vaccine because the prime-boost vaccine is administrated in a single dose. Therefore, this delivery system makes vaccines more accessible to a larger population and brings great benefit to remote areas that have minimal access to medical services. In the present work, a novel synthesis method was first developed to synthesise mesoporous silica spheres with pore sizes tailored to match specific molecules or applications. This new method offers greater control of pore size by using inexpensive commercial silica colloids and a simple electrolyte in the presence of a temporary polymer hydrogel network. Silica colloids were used as a feedstock for the mesoporous silica because the resultant pore size of the mesoporous silica is not limited by the molecular size of the organic template. Using this method, mesoporous silica spheres at the sub-micrometer and micrometer scale (0.50 to 1.60 μm) with a tailored pore size (14.1 to 28.8 nm) were obtained. In order to investigate the potential application of these mesoporous silica spheres to provide a delivery platform for biomolecules, adsorption studies of a model protein (bovine serum albumin, BSA) onto the mesoporous silica spheres were performed by employing real time in situ measurements. The adsorption isotherm obtained fitted the Langmuir model and high adsorption capacities (up to 71.43 mg BSA/ml adsorbent) were observed. It was found that the conformation of the BSA molecules remained intact after the in vitro release kinetics studies. In the current study, a new and improved microencapsulation method, which shields the pDNA molecules from deleterious conditions and enables a narrow microsphere size distribution, was developed. In this method, the pUC19 plasmids are condensed with polyethylenimine (PEI), and then embedded into poly(lactide-co-lactide) (PLGA) using a 40 kHz ultrasonic atomization system. Biodegradable polymer microspheres with volume weighted mean diameters (D[4,3]) of 6.0 – 15.0 μm were obtained and 95 – 99% of the encapsulated pUC19 plasmids exhibited zero order release kinetics over the 30 day in vitro delivery studies. The use of ultrasonic atomization for the production of biodegradable polymer microspheres containing pDNA molecules is a new application. This method has several distinct advantages including the ability to control particle size; the ability to self clean; and it does not require elevated temperatures and phase separation inducing agents. In order to study the feasibility of ultrasonic atomization system in microencapsulating the prime-boost vaccines, the system was then extended to encapsulate both pEGFP-N1 plasmids and mesoporous silica spheres loaded with BSA into PLGA. The feeding system was modified by feeding the feedstocks into the nozzle of the atomizer through the dual-concentric-feeding needles. This fabrication technique produced composite microspheres with D[4,3] ranging from 6.0 to 34.0 μm, depending on the conditions during the microsphere preparation, such as polymer concentration and volumetric ratio. The in vitro release profiles obtained over 40 days showed that pDNA and protein have different release kinetics. Protein appeared to follow quasi-zero order release kinetics with a minimal initial burst rate. These release profiles showed an effective method to enhance immune responses by facilitating different delivery regimes between the pDNA prime and protein boost through advanced particle design. This technique has the potential for aseptic manufacturing and easy scaling-up for industrial applications. The development of enhanced vaccine delivery system is particularly important to combat a host of current and emerging infectious diseases in areas with limited medical services.