Kitosaani-pDNA-nanopartikkelien kehittäminen ja karakterisointi

Chitosan has been explored as a non-viral vector for gene therapy due to favourable characteristics such as good biocompatibility and biodegradability. The aim of this thesis was to prepare and characterise chitosan-plasmid DNA (chitosan-pDNA) nanoparticles, using 17 different types of chitosan, in order to evaluate which chitosan yields the most favourable nanoparticle characteristics in terms of size and zeta potential. Also size stability and correlations between chitosan properties such as molecular weight (Mw) and degree of deacetylation (DD), and particle properties were assessed in this study. Both basic chitosan and chitosan salts and derivatives were tested. The particles were prepared by complex coacervation. Size and zeta potential of particles were determined by dynamic light scattering (DLS) and Laser Doppler velocimetry, respectively. Integration of pDNA with chitosan was assessed by agarose gel electrophoresis and morphology of prominent samples was observed using field emission gun scanning electron microscope (FEG-SEM). With a fixed chitosan to pDNA weight ratio 4:1 most chitosan types, excluding carboxymethylchitosan and chitosan oligosaccharide, integrated with pDNA to form nanoscale particles. The size of the remaining chitosans ranged from approximately 100 nm to 180 nm and the zeta potential was relatively stable ranging from +20 to +30 mV. Smallest particles were achieved with chitosan 85/25 (101.2 ± 0.9 nm) and chitosan hydrochloride (100.3 ± 1.3 nm). Statistical analysis indicated that for most samples particle size increased significantly during the first week and then remained relatively stable. No clear correlations between chitosan and nanoparticle properties were detected. However, it appears that high DD and high Mw chitosans yield more stable complexes indicated by higher zeta potentials. According to FEG-SEM images particles were spherical and their size ranged from 20-200 nm. Smaller particles appeared to have a more even structure. In many cases, however, the particle surface was not smooth suggesting possible aggregation. Although chitosan 85/25 and chitosan hydrochloride yielded good results in terms of size, it was noted by other researchers in the same project that functionalisation of the particles failed due to sedimentation. When the weight ratio was changed, functionalisation properties improved. Therefore, future research should focus on exploring the effects of different chitosan:pDNA weight ratios on particle parameters and also functionalisation possibilities should be considered. /Kir1