graphpad.com). The data were not normally distributed and hence statistical significance was tested using the Kruskal–Wallis test. When the results were significant, differences among the individual medians were examined using the Mann–Whitney test. Significant effects were declared when P < 0.05. The incorporation efficiency of PTd in the MPs was estimated to be around 78% for PTd and 95%
for CpG and HDP. Previous studies showed that particles less than 10 μm are preferentially taken up by APC [12], [15] and [16]. As such, SEM of MPs that comprised of PCEP with CpG ODN, and IDR-1002 was performed to ensure that the resulting size of the particles was compatible with uptake into APC to ensure that an effective dosage of antigen would be processed. Our previous studies of encapsulated CpG ODN using the same methodology #Libraries randurls[1|1|,|CHEM1|]# not only showed that the MPs generated were less than 10 μm, but also revealed 99% uptake into murine macrophages [12] and [15]. Indeed, the addition of IDR-1002 into the MP was consistent with these previous findings revealing particles ranging in size from 0.5 to 5 μm in diameter (Fig. 1A and B). At higher magnification (20,000×), a close inspection of the surface of these MP revealed that it was not smooth; instead, the surface of these MP seem to be composed
of smaller nanoparticle structures (Fig. 1C). To assess the efficacy of MP formulation, we compared the levels of the pro-inflammatory cytokines Proteases inhibitor TNFα, IL-6 and IL-12p40 in murine J774 macrophages treated with CpG ODN-IDR (AQ), PCEP-CpG ODN-IDR (SOL) and MP co-encapsulating PCEP-CpG ODN-IDR. Other than measuring pro-inflammatory responses, we also looked for the chemokine MCP-1, a chemotactic agent for monocytes/macrophages, T cells, NK cells, and neutrophils, since
it was previously shown that both CpG ODN and the IDR-HH2 alone enhanced MCP-1 Levetiracetam production [17], while their complexes demonstrated a synergistic increase in production [11]. The induction of MCP-1 was strongest with the SOL formulation compared to the MP formulation (Fig. 2A) co-encapsulating CpG ODN-IDR complexes or CpG ODN and HDP delivered in uncomplexed MP. The release of pro-inflammatory cytokines TNF-α and IL-6 was significantly higher in MP treated macrophages than AQ or SOL formulation treated groups (Fig. 2B and D). The IL-12p40 levels were two-fold higher in the MP than SOL or AQ formulation treated groups (Fig. 2C). LPS was used as a positive control to demonstrate the viability of the cells. Based on these results, we conclude that the MP delivery induced higher levels of pro-inflammatory cytokines in mouse macrophages.