Download Annette Vinther Heydenreich

Abstract nr. F9
Ph.d.-studerende
Annette Vinther Heydenreich
Institut for
Farmaci
Studiestart
1. november 1998
Vejledere
Henning Gjelstrup Kristensen og Anders Fomsgaarda
Titel på forskningsprojekt
Mucosal DNA Vaccine Delivery with Cationic Solid Lipid
Nanospheres
a
Department of Virology, Statens Serum Institut, Copenhagen
Genetic immunization (DNA vaccines) has the potential to both produce neutralizing antibodies (humoral
immune response) and cytotoxic T-cells (cellular immune response), which is believed to be essential in viral
infections like HIV. In order to stop the viral replication at the site of entry, mucosal immunity with high
levels of sIgA antibodies can obtained by vaccination at the musosal sites. Efficient delivery of genetic
vaccines requires a drug delivery system able to stabilize/ protect DNA against nucleases, to facilitate
cellular uptake and endo/lysosomal release once taken up and to mediate cell-specific targeting. The purpose
of this study is therefore to investigate Solid Lipid Nanospheres (SLN) as a particulate drug delivery system
for mucosal genetic immunization.
Solid Lipid Nanospheres (SLN) combine the advantages from liposomes (e.g. biocompatible compounds)
and solid drug carriers as polymeric nanoparticles (e.g. prolonged circulation time, protection of the drug)
without having the disadvantages of viral delivery systems. Mucosal (nasal, oral, rectal) administration of
particles are taken up by the M-cells and are transported directly to the dendritic cells in the underlying
immune tissue.
SLN were obtained by diluting warm microemulsions (o/w) composed of different fatty acids or triglycerides
(all solid at room temperature), cationic lipids, nonionic surfactants and cosurfactants in cold water. After
washing the SLN by dialysis, particle size distribution and zetapotential was measured. The resulting
particles were between 100 and 500 nm in size (PI~0.3) and showed zetapotentials between +10 and +35 mV
at pH 7.4. Physico-chemical properties of SLN were strongly dependent on the composition. Additionally,
morphology was examined by scanning electron microscopy (SEM) showing spherical particles.
The DNA was either incorporated in the SLN during the preparation or complexed to the surface at different
charge ratios after the preparation. The latter was having little effect on the particle size. The physical
stability of the SLN-DNA complexes was dependent on the ionic strength of the solution, the loading time
and the zetapotential of the particles, indicating more than an electrostatic interaction. The extracted DNA
and the complexation were analyzed by agarose gel electrophoresis, showing no degradation and change in
structure. The complexes were further characterized by SEM and zetapotential measurements. The SLN were
able to protect the DNA against degradation by DNase I. Cytotoxicity (MTT test) and transfection efficiency
were tested in vitro using pCMV-EGFP in various cell-lines (HEK 293, CPH 54A and NR6). Uptake,
localization and co-localization studies were performed using the lipid soluble fluorescent dye 4,4-Difluoro1,3,5,7,8-penta-methyl-4-bora-3a,4a-diaza-s-indacene (BODIPY492/515), pGeneGrip Rhodamine/GFP and
LysoTracker Red DND-99.
The cells studies showed uptake of SLN and DNA in various fibroblast cell lines and in immature dendritic
cells, preferentially in the cytoplasm. No transfection was observed. In the literature no correlation between
in vitro and in vivo transfection is observed, and in vivo immunizations in mice are ongoing. Positive results
are still to come.
Conclusions. Cationic SLN may serve as potential particulate delivery systems for plasmid DNA in regard
to protection of DNA and their sizes suggest an uptake by M-cells. But they have to be optimized in order to
escape the lysosomes and thereby deliver DNA to the site of action.