## 4 Discussion  
In this study, we have used two phagocytosis cell lines that give a comprehensive indication of the interaction between particles and immune cells of the native immune system. THP-1 and RAW 264.7 cells have both been extensively used to study macrophage functions, mechanisms, signaling pathways and drug transport [19; 20]. THP-1 cells are a human monocytic suspension cell line, whereas RAW 264.7 cells are an adherent mouse macrophage cell line. Adherent cells can be assumed to be more efficient in their phagocytosing capability due to their capability of moving and adhering to sedimented particles before phagocytosis [32]. Once nanoparticles enter a physiological environment, the surface of nanoparticles is rapidly covered by proteins to form a corona. Adsorption of proteins to the surface depends on the particle's surface charge, and high amounts of adsorbed protein presuppose an increased cell interaction due to opsonins such as IgG and complement factors [32; 33]. A positive surface charge, as for Alhydrogel at pH 7, increases protein adsorption at pH 7, probably increasing the association between cells and particles. The opposite applies to negatively charged particles such as the SA-MIPs [14], which exhibit low protein adsorption after incubation in a serum-containing culture medium (Figure S3). This could explain the difference between SA-MIPs and Alhydrogel, as observed by flow cytometry (Figure 1). While all the cells became associated with Alhydrogel upon incubation with cells, only 20-30% of the cells became associated with SA-MIPs upon incubation. The SA-MIPs have a specificity for SA expressed by the target cells and the present results correspond well with previously reported SA-MIP binding results based on the expression of SA by various cancer cell lines [4; 6; 7; 14; 34]. In our study, the focus was not on the binding efficiency of SA-MIPs to its targets, but on the cellular uptake and possible interference of the SA-MIPs with phagocytosis cells. The association between cells and particles is a pre-requisite for phagocytosis and it is well-documented that the reference particle Alhydrogel is phagocytosed by viable cells [35]. In accordance with flow cytometry, the fluorescence microscopy images showed a limited number of cells associated with the SA-MIPs; although, the images also indicated that the associations of SA-MIPs were both on the cell surface and intracellularly (Figure 2), implying modest phagocytosis of the SA-MIPs by the macrophages; although, the particles had a specificity for the phagocytosis cells. Cytokines are synthesized and released in response to activation of pattern recognition receptors or by activation of the inflammosomes, something which can occur upon phagocytosis of particles [17]. Although a restricted number of inflammatory cytokines were investigated in this study, the investigated cytokines, IL-1\(\upbeta\), IL-6 and TNF-\(\upalpha\) are major inflammatory cytokines and are expected to be secreted when inflammation is triggered. THP-1 cells do not produce the pro-form of IL-1\(\upbeta\) and formation of the NALP3 (NLR family pyrin domain containing three) inflammasome unless pre-stimulated with LPS [36; 37]. Upon activation of the inflammosomes, caspase 1 is activated, which cleaves the pro-form of IL-1\(\upbeta\) into IL-1\(\upbeta\), which becomes secreted into the medium [38; 39]. After pre-stimulation with LPS, THP-1 cells were induced to assemble the NALP3 inflammasome and to synthesize pro-IL-1\(\upbeta\). THP-1 cells pre-stimulated with LPS and cultured with SA-MIPs only showed a minor secretion of IL-1\(\upbeta\), which means that the inflammosomes and thereby the caspase 1 was not activated by the SA-MIPs. Al-adjuvants are known inducers of the inflammosomes [40; 41; 42], and as expected, a high IL-1\(\upbeta\) secretion was observed in the presence of Alhydrogel. Several other inflammatory cytokines are also induced and secreted by monocytes and macrophages through activation of the NF-kappaB signaling pathway [33; 43].