(175c) Inorganic-Organic Composite Particle as a Staged Delivery Platform for Plasmid DNA-Based Biopharmaceuticals

Infectious diseases such as SARS, influenza and bird flu may spread exponentially throughout communities. In fact, most infectious diseases remain major health risks due to the lack of vaccine or the lack of facilities to deliver the vaccines. Conventional vaccinations are based on damaged pathogens, live attenuated viruses and viral vectors. If the damage was not complete, the vaccination itself may cause adverse effects. Therefore, researchers have been prompted to prepare viable replacements for the attenuated vaccines that would be more effective and safer to use.

DNA vaccines are generally composed of a double stranded plasmid that includes a gene encoding the target antigen under the transcriptional directory and control of a promoter region which is active in cells. Plasmid DNA (pDNA) vaccines allow the foreign genes to be expressed transiently in cells, mimicking intracellular pathogenic infection and inducing both humoral and cellular immune responses. Currently, because of their highly evolved and specialized components, viral systems are the most effective means for DNA delivery, and they achieve high efficiencies (generally >90%), for both DNA delivery and expression. As yet, viral-mediated deliveries have several limitations, including toxicity, limited DNA carrying capacity, restricted target to specific cell types, production and packing problems, and high cost. Thus, nonviral systems, particularly a synthetic DNA delivery system, are highly desirable in both research and clinical applications.

At present, DNA immunization can be achieved using intramuscular or intradermal injection, gene gun administration, and etc. The direct injection of naked plasmid DNA is possible, but relatively few cells take up the DNA (1-3%) due to the high molecular weight and polyanionic nature of the nucleic acids, leading to reduced expression of the encoded protein. Moreover, free pDNA deliveries are also hindered by their instability in biological fluids due to the degradation by the endonucleases. Efficiency issues surrounding the delivery of DNA vaccines can be overcome by administering larger doses, but this can cause the unwanted side effects of toxicity and multi-drug resistance, as well as increased cost per dose.

One of the problem with vaccinations affects global health is unsafe injection practices. The World Health Organization (WHO) estimates that over 12 billion injections are administered annually and up to 30% of these injections are unsafe. The transmission of bloodborne pathogens from patient to patient with unsterilized needles has been recorded for over half a century. These unsafe injection practices are associated with substantial morbidity and mortality, particularly from hepatitis B, hepatitis C and HIV infections, especially in poorer and developing countries. Currently, oral administration is another popular route for drug/vaccine delivery. However, oral drug delivery is hampered by the low mucosal permeability of drugs, and lack of stability in the gastrointestinal (GI) environment, results in degradation of the drugs compound prior to its absorption.

As a result, there are an increasing number of drugs/vaccines being administered to humans via the nasal route. The nasal route is an important arm of the mucosal immune system since it is often the first point of contact for inhaled antigens. After intranasal immunisation, both humoral and cellular immune responses can occur. Intranasal administration will greatly assist progress of health programs around the world since nasal vaccines offer non-invasive administration, are easily accessible for a larger population, and do not require trained persons for administration. Easier and safer administration in addition to a decreased total cost of treatment would represent a major breakthrough for global vaccination programs, especially for developing countries. The use of particulate delivery systems for administration of DNA through the nasal route is not a strategy that has attracted a large volume of research as the advent of DNA vaccination is relatively new.

Another problem associated with DNA vaccines is there is an urgent need to develop their potent and efficient before they can be used effectively in humans, since the failure to elicit antibodies against antigens when used in human has been reported recently. The development of an effective carrier system may be the key element in improving and homogenizing the overall immune response to DNA vaccines. One of the today's trends is to associate DNA-based immunization with other immunization approaches, such as vectors or antigens, as part of prime-boost (mixed) immunization regimes. DNA prime-protein boost immunization involves priming with DNA vaccines and boosting with protein or recombinant protein. These strategies aim to augment immune responses to pathogens. Thus, there needs to be a particulate delivery system that enables staged delivery of DNA prime-protein boost in the nasal tract. Improved staged delivery of vaccine can be achieved by employing a composite of inorganic mesoporous silica and biodegradable polymer-based particulate delivery system. These particulates are able to maintain sustained vaccine release over a period of time to specific sites for prime-boost vaccination.

Inorganic host materials, mesoporous silica is a relatively benign material in terms of biocompatibility and versatile in terms of the variety of chemical and physical modifications that are available. It possesses high specific surface areas, high specific pore volumes, and well-ordered pore structures adjustable within the range of 2 - 50 nm. Great progress has recently been made in the utilization of mesoporous silica spheres as a nonviral gene delivery vector, as well as its good immobilization capacity for various biomaterials. Mesoporous silica exhibits its diversity and potential applications in many facets of biological science. Methods such as solution growth and aerosol self assembly have been developed to synthesize mesoporous silica spheres. Organosilicates such as tetraethylorthosilicate (TEOS) are used in these syntheses, and particularly organic templates are essential for generating the mesoporous structures of silica spheres. However, organosilicates are expensive. This drawback has hindered the usage of these alkoxide precursors in large-scale application. Economic considerations have aroused the usage of inexpensive inorganic silica as a starting material for mesoporous silica and further increase its realistic applications. Moreover, larger pore is demanded for advanced application of the mesoporous materials in biotechnology due to the encapsulation of biomolecules in these materials.

In this current study, a novel synthesis of mesoporous silica spheres has been developed by using a simple electrolyte and inexpensive commercial inorganic silica colloids. This new method will offer a great flexibility in tuning or tailoring the pore size of the mesoporous silica spheres to match specific molecules or applications, and to produce large quantities of mesoporous silica spheres for potential use in bio-nanotechnology, drug delivery and inorganic adsorbent applications. Mesoporous silica spheres at the sub-micrometer and micrometer scale (0.5 to 1.6 ìm) with a tailored pore size (14.1 to 28.8 nm) has been obtained. The influences of synthesis conditions including solution composition and calcination temperature on the formation of the mesoporous silica spheres were systematically investigated. Crosslinked polyacrylamide hydrogel was used as a temporary barrier to obtain dispersible spherical mesoporous silica spheres. Adsorption of protein onto these particles and the in vitro release profile will be presented. The adsorption isotherm fitted the Langmuir model; a very high adsorption capacity (71.4 mg/ml adsorbent) has been obtained. The mesoporous silica spheres released ~21 % of protein loaded in the initial burst period and starts to plateau subsequently. Results obtained from the automated electrophoresis gel revealed that conformation and size (in kDa) of protein were the same before and after the adsorption studies. This indicates that biopharmaceuticals can retain intact upon desorption from a mesoporous silica spheres delivery platform.

The embedding of biopharmaceutical compounds into biodegradable polymer microspheres has gained considerable interest over the last decade. Vaccines can be released from such microparticles in a sustained and controlled fashion, while non-release biopharmaceuticals are protected from rapid in vivo degradation. Adhesive properties of biodegradable polymer make them suitable for transmucosal delivery applications and prolong the contact time of biopharmaceuticals with the nasal surface. Moreover, these biodegradable polymers can degrade to toxicologically harmless products. In the present work, several candidates (i.e. PLGA and etc.) will be studied for their suitability in the encapsulation of pDNA, which mainly based on their degradation mechanism and profile, porosity, and network structure. Furthermore, the selection of a suitable microencapsulation technique for biopharmaceuticals is crucial due to sensitivity of these biopharmaceuticals towards processing conditions. As a result, several characteristics can be used to evaluate the suitability of a chosen method, include molecular interaction properties between biopharmaceutical and polymer, stability during processing and etc. Therefore, a new and improved process, shielding the antigen from deleterious conditions has to be developed. In this study, a microencapsulation method using ultrasonic atomization to synthesize biodegradable polymer microspheres that encapsulate both the pDNA and the mesoporous silica spheres loaded with protein for nasal delivery application will be presented. The in vitro staged delivery profile for pDNA and protein from the resulting composite particle will be presented. The use of ultrasonic atomization for the production of biopharmaceuticals containing biodegradable polymer particles is a comparatively new application. The advantages of this atomization are the possibility of particle size control, and the fact that it does not require elevated temperature and phase separation inducing agents. This novel technology appears to have the potential for aseptic manufacturing and easy up-scaling for industrial applications.