CONSECUENCIAS DE FALLA

The use of delivery systems for CpG ODNs to enhance the ODNs activity is currently one of the immunological topics that are discussed e.g. to be utilized as adjuvant formulation or as all-in-one vaccine formulation with a co-delivered antigen. In this context, our partners at University of Alberta and others have shown that the immunogenicity can be increased by its delivery in biodegradable

micro- or nanoparticles (Singh et al. 2001; Diwan et al. 2002; Diwan et al. 2004). In this study we used for the first time cationized gelatin nanoparticles as carrier for CpG ODNs. Gelatin offers several advantages as a biomaterial for preparation of colloidal drug carriers. In the current study, we set out to demonstrate that CpG ODN could be efficiently loaded onto cationized gelatin nanoparticles and that these formulations could effectively be internalized by MDCs. A loading ratio between 10:1 and 20:1 (w/w) of nanoparticles to CpG ODN was found to be favorable since the formulations remained stable within physiological media and had a slightly positive net charge. These positively charged nanoparticles have been reported to be better phagocytosed by MDCs and macrophages than neutral or negatively charged particulate formulations (Roser & Kissel 1993; Thiele et al. 2001; Foged et al. 2005). In agreement with this, MDCs internalized the positively charged CpG ODN nanoparticles more efficiently than the non-cationized gelatin nanoparticles, which have a partially negative ζ potential (see Chapter II - 3.1). CLSM studies showed that the uptake of nanoparticles was inhibited by treatment of MDC cultures with Cytochalasin B, an inhibitor of intracellular actin-myosin polymerization, an essential step in phagocytosis (Davis et al. 1971). Thus, the observed uptake of the cationized gelatin particles by MDCs could be assumed to be driven by active phagocytosis, and neither due to mere adherence of the particles to cell surfaces, nor due to pinocytosis. Upregulation of MHC and co- stimulatory molecules on the MDC surface improves the ability of MDCs to activate and prime T cells. The results of our study indicated that CpG ODN loaded onto the gelatin nanoparticles superiorly upregulates MHC-II and CD86 molecules. These data also indicated that MDCs were in their immature state at the time of phagocytosis and CpG that ODN loaded onto the cationized nanoparticles efficiently induced MDC maturation. Similar to soluble CpG ODN which promotes the secretion of TH1 polarizing cytokines and cytotoxic immune response

by MDCs (Chu et al. 1997; Krieg et al. 2000), CpG-GNPs induced the secretion of IL-12p70, IL-6, and TNF-α. However, the extent of secretion of IL-12p70 and TNF-α was amplified in comparison to soluble CpG ODN treatment. No significant difference in the extent of cytokine secretion was observed for the secretion of IL-6. Since IL-6 is discussed to have adverse effects on the TH1

differentiation (Diehl & Rincon 2002), this circumstance would favor a bias towards T 1 immune response even more. Regarding particle size, overall CpG

slightly higher immune stimulation than loaded on ZWCa01, having a mean size of 135.3 nm. This finding is in agreement with Foged et al. (Foged et al. 2005), who tested the phagocytosis activity of various polystyrene particles in a diameter range between 0.04-15 µm and found that particle diameters around 0.5 µm and slightly below were optimal for uptake into MDCs. Consequently, our system might be advantageous compared to already established microparticulate carrier systems. The absence of cytokine secretion following the treatment of MDC cultures with plain nanoparticles, or control nonstimulatory ODN, indicated a basic biocompatibility of the nanoparticles and the preparation process, as any endotoxin contamination would have triggered cytokine secretion by MDCs.

Investigating the reaction kinetics of CpG-GNP uptake into MDCs, it was noticed that the formulations were quickly internalized within 20 min. The amount of the internalized CpG-GNPs can be influenced by the administered concentration. Congruent to the cell toxicological data we generated for cationized gelatin nanoparticles in gene therapeutic experiments, even huge amounts of CpG-GNPs (100 µg per 2 x 104 _{MDCs) did not cause significant cell death. However, cell}

clustering occurred at this high amount of administered CpG-GNPs. So it seems to be advisable to reduce the amount of CpG-GNPs below this value.

Cytokine secretion can be detected within 3-12 h after CpG-GNP administration. In comparison to soluble CpG ODN, no retarded or sustained cell activation could be observed. Thus, CpG ODNs seem to be still attached onto the nanoparticles when interacting with TLR9. This apparently induces enhanced but not prolonged activation of the immune system.

Interestingly, polymers known for their good properties in non-viral gene delivery due to quick endosomal release (e.g. PEI) lead to less activation when administered as polyplex with CpG ODNs than soluble CpG ODN alone (Ilardia et al. 2006; unpublished data Dr. Dr. Bourquin). But since TLR9 is located within the endosome, quick endosomal release is undesirable in this case. In pDNA transfection experiments with cationized gelatin nanoparticles, the gene expression occurred with certain delay (see Chapter II). Due to the present CpG ODN data it appears obvious that this delay was caused by sustained endosomal release in comparison to PEI polyplexes. Summarizing, it seems that these properties of cholamine-modified cationized gelatin nanoparticles that are rather unfavorable for gene transfection, are ideal for CpG ODN delivery.

CpG ODNs are reported to be less toxic than other investigated adjuvants (Weeratna et al. 2000). However, other studies have reported dose-dependent systemic adverse effects for CpG ODN (Klinman et al. 1996; Lipford et al. 2000; Heikenwalder et al. 2004). Significant dose reduction would be expected to minimize the risk of side effects of this new class of immune adjuvants. Nanoparticulate CpG ODN delivery decreases the effective dose needed to generate or increase an immune response in the present in vitro data.

The present data is a promising first step towards the application of cationized gelatin nanoparticles in the field of immuno therapeutics. Even though, correlation of the murine in vitro data with humans or non human primates seems, due to the previously mentioned different cellular distribution of TLR9, doubtful. So, further experiments with primary human B cells and plasmacytoid dendritic cells (PDCs) were indispensable in addition to the present data.