Microparticular and Nanoparticular DNA Delivery Systems as Adjuvants for DNA Immunization
In this dissertation different microparticular and nanoparticular DNA carrier systems were developed, with the aim to create an efficient adjuvant system for DNA vaccination. Their suitability was investigated by physico-chemical parameters, such as particle size, z-potential and encapsulation eff...
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|In this dissertation different microparticular and nanoparticular DNA carrier systems were developed, with the aim to create an efficient adjuvant system for DNA vaccination.
Their suitability was investigated by physico-chemical parameters, such as particle size, z-potential and encapsulation efficiency. Further, the systems were studied in-vitro for DNA stabilization and DNA bioactivity after encapsulation and release, as well as for gene delivery.
We investigated modified double emulsion methods and spray drying techniques for DNA microencapsulation. Firstly, DNA was complexed with polyethylenimine (PEI) 25 kDa. We further studied the possibility to encapsulate lyophilized DNA and lyophilized DNA / PEI complexes in the presence of lyoprotectants. The microparticles were formulated using i) a modified double emulsion technique (W/O/W), ii) a solid in oil in water method (S/O/W), iii) a water in oil spray drying technique (W/O) and iv) a solid in oil spray drying technique (S/O). DNA release from particles prepared with double-emulsion methods, in contrast to spray drying techniques, resulted in constant DNA release and relatively low initial burst effects. The complexation with PEI substantially retarded the DNA release for all preparation techniques.
In Chapter 4, we adsorbed DNA onto the surface of microparticles. We developed a cationic microparticular system by the incorporation of different amounts of the cationic molecules, PEI or CTAB into the polyester matrix. PEI 10% microparticles exhibited the most promising characteristics, such as a small particle size, a high z-potential of + 47 mV, a high DNA adsorption efficiency for a theoretical loading of 1% over the physiological pH range. The mechanism of gene delivery was studied by confocal microscopy and revealed diffuse fluorescence of DNA and PEI in the cytoplasm of non-phagocytic L929 fibroblasts. This was attributed to polyplex formation after PEI release from the particle. The efficient gene transfer of RG 502H+PEI 10% microparticles was confirmed by luciferase transfection. The challenge experiments with a lethal dose of the pathogen in challenge experiments in mice demonstrated that the formulation had an adjuvant effect.
In Chapter 5, a new polymeric system was designed, consisting of poly (vinyl-alcohol) coupled with diamines, such as diethylaminopropylamine (DEAPA), (DMAPA) or (DEAEA). The amphiphilic properties allowed the formulation of DNA nanoparticles by a modified solvent displacement technique without the use of shear forces. DNA nanoparticles exhibited positive z-potentials up to +42 mV. The gene delivery of the nanoparticles was assessed in L929 mouse fibroblasts, which demonstrated high transfection efficiencies, comparable to PEI 25kDa/DNA complexes at a nitrogen to phosphate ratio of 5.
In Chapter 6 we chose one representative polymer, P(26)-10, of the new class of amine-modified polyesters to investigate the influence of several process parameters on the nanoparticle formation.
In Chapter 7, DNA nanoparticles with amine-modified polyesters were further characterized using two classes of polymers (DEAPA /DEAEA) with different amounts of amine modifications. The nanoparticle x-potentials and sizes were dependent on the N/P ratio. Atomic force microscopy confirmed the small particle sizes. DNA stability during the encapsulation process and release over nine day was demonstrated by electrophoresis, as well as DNA protection from enzyme degradation in dependence of the N/P ratio.
The amount of cellular uptake of an efficient candidate P(68)-10, DNA nanoparticles was shown to be dependent on the N/P ratio of the formulation by flow cytometry. The mechanism of cellular uptake was followed by confocal microscopy and exhibited endocytotic uptake of the particles. The very efficient gene delivery of the P(68)-10 polymer was demonstrated by in-vitro transfection assays in four cell lines compared to PEI / DNA complexes at equal N/P ratios.