Construcción de sentido

To track the in vivo fate of CpG-GNPs, mice were injected with fluorescence- labeled CpG-GNPs. After 4 hours, the mice were sacrificed and their major organs were resected. Surveying selected tissue samples that were obtained from animals receiving intravenously administered CpG-GNPs under a confocal laser scanning microscope, it was possible to localize fluorescence-labeled CpG-GNPs in addition to blood cells also in liver, spleen and kidney (Fig. 5A). These data correlated nicely with other biodistribution data that were obtained with gelatin nanoparticles (Kaul & Amiji 2004; Zillies et al. 2005). Subcutaneously administered CpG-GNPs could not be detected in those tissues (Fig. 5B), thus the particles were obviously not further processed by the systemic bloodstream, at least not above the visually detectable limits.

blood cells liver spleen kidney

Fig. 5: CLSM images of various tissues. Blue: cell nuclei; Green: fluorescence-

labeled CpG-GNPs. A: intravenous injection; B: subcutaneous injection

Consequently, CpG-GNPs were only systemically distributed via the blood stream following intravenous injection instead of subcutaneous. These findings agree with those previously described for other colloidal drug delivery systems, such as solid lipid nanoparticles (Reddy et al. 2005) and liposomes (Oussoren et al. 1997). Those references describe that s.c. administered colloidal carriers frequently remain at the injection site and are partially transferred via the lymphatic system to the draining lymph nodes. In detail, the carriers are taken up at the site of injection by local phagocytotic cells such as immature DCs. Following this uptake the cells transform into their mature state and are transferred to the lymph nodes where they start to induce a local immune response, such as e.g. the TH1/CTL-type response that is

activated by CpG ODNs.

Due to this theoretical background, it was decided to further investigate tissue samples of the site of injection and the draining lymph nodes. Since the subcutaneous injections were conducted into the footpads, the next draining lymph nodes were located in the popliteal fossa and the inguinal region. However, confocal laser scanning microscopic analysis 4 h past injection revealed only a massive amount of nanoparticles at the site of injection. No nanoparticles could be identified in the draining lymph nodes (Fig. 6).

A

Fig. 6: CLSM images of tissue samples at 4 h past subcutaneous injection; A: tissue

at the site of injection; B: tissue of the popliteal lymph node

Even though the nanoparticles could not be visually detected under the microscope after 4 h, it was decided to analyze the tissue of the popliteal and inguinal lymph nodes by more sensitive FACS analysis at various time points. In Fig. 7, a typical quadrant analysis of the cells derived from popliteal (Fig. 7A) and inguinal (Fig. 7B) lymph nodes is shown.

Fig. 7: 2-color FACS analysis of cells that were derived from the draining lymph

nodes being closest to the site of CpG-GNP injection. Sacrificed: 18 h post injection;

y-axis: FL-1 fluorescence (Alexa FluorTM _{488 labeled CpG-GNPs); x-axis: FL-2}

fluorescence: Allophycocyanin (APC) anti-mouse F4/80 antigen; A: popliteal lymph node; B: inguinal lymph node

In the displayed two-color analysis macrophages have been additionally stained with F4/80 APC. They can be identified in the upper and lower right quadrants. Cells that have taken up nanoparticles are located in the upper quadrants. The results show that nanoparticles could be located within the cells of lymph nodes. In total, more nanoparticles could be located after 18 h in the popliteal lymph nodes that are located closest to the site of administration (Fig. 7A), whereas only a few

A B

apparently taken up by various distinct cell-types, such as macrophages and other phagocytotic cells (DCs, etc.).

Based on these histological results, the conclusion can be drawn that s.c. administered CpG-GNPs do not cause a systemic immune activation since they are, in contrast to soluble CpG ODN, not removed via the blood stream from the site of injection. But, as a certain movement towards the draining lymph nodes could be observed, it was potentially possible that an activation of the immune system by CpG-GNPs occurs. However this would only happen within a regionally restricted area encompassing the injection site.

3.1.4 Summary

The results of these first experiments to characterize the systemic immunogenic activity of CpG-GNPs revealed significant differences to soluble CpG ODN. Whereas soluble CpG ODN induces a systemic secretion of TH1/CTL-polarizing

cytokines subsequent to both types of administration, CpG-GNPs did only cause a systemic immune response after intravenous injection.

Analyzing the systemic cytokine levels that were induced with i.v. administered CpG-GNPs, in vitro results indicating the size of the applied gelatin nanoparticles as significant factor on the immunogenic activity of CpG-GNPs were confirmed. The ideal nanoparticle size to excite the secretion of enhanced levels of IL-12p70, the key cytokine to promote TH1 differentiation, is apparently ~300nm. Observing the

maturation or activation levels of myeloid DCs (MDCs), plasmacytoid DCs (PDCs), B cells, and CD8+ _{cells as second important benchmark aside from}

cytokine secretion revealed that CpG-GNPs led to comparable enhanced numbers of activated cells as soluble CpG ODN.

Searching for possible explanations, why subcutaneous administration did not result in any detectable systemic cytokine levels, CLSM analysis gave valuable insights. S.c. administered CpG-GNPs are in contrast to i.v. injected CpG-GNPs not biodistributed via the bloodstream, thus remaining basically at the site of injection. Nevertheless, FACS analysis revealed that some nanoparticles are also transported to the closest draining lymph nodes.

Consequently, it might be possible that CpG-GNPs are still able to induce an immune response. But due to the lack of systemic distribution, no systemic cytokine secretion occurs. If this potential local immune reaction would be

sufficient to e.g. boost the immune response against a co-administered antigen in comparable manner to soluble CpG ODNs, but without systemic side-effects, this would be a highly auspicious application.

3.2

CpG-GNPs as immunization adjuvant for chicken egg