In the clinics, many inflammatory diseases are handled with GCs, if necessary with high doses and/ or for a long period of time, which often induces severe side-effects and resistance. In theory, targeted delivery of GCs could circumvent these issues (Vandewalle et al., 2018). The type of carrier system, however, is essential for the outcome. For example, PEG-LPs have the disadvantage to provoke an unwanted effect, namely the activation of the complement system (Sercombe et al., 2015). Moreover, the characteristics of NPs, with respect to shape, size or stability influence the interaction with the target cell and needs to be evaluated (Kuhn et al., 2014). Hence, we analyzed the cell type specificity, toxicity, cellular uptake mechanism and intracellular fate of IOH-NPs, which were available to us in this project.
Due to their hydrophobic structure, GCs can easily pass the cell membrane, where they bind to the cytosolic GR (Strehl and Buttgereit, 2013). GCs
DISCUSSION
120 encapsulated in nanoparticles, like IOH-NPs, need to enter the cell via an active process called endocytosis. The endocytic capacities vary between cell types, thus contributing to the IOH-NPs cell-specificity (Behzadi et al., 2017). To get a good impression of the specificity of IOH-NPs, we selected cell lines representing macrophages, T and B lymphocytes, fibroblasts, epithelial cells and myoblasts. Longer incubation times increased the uptake in all cell lines, which indicates an increasing accumulation of IOH-NPs within all cells. MH-S cells, resembling macrophages, were most efficient in taking up IOH-NPs.
Furthermore, L929, resembling fibroblasts were somewhat weaker on the long-run as MH-S cells while LA-4 cells and C2C12 cells, resembling epithelial cell and myoblasts, respectively, took up IOH-NPs with only moderate efficiency. In contrast, the cell lines WEHI7.1 and WEHI231, representing T and B lymphocytes hardly took up any IOH-NPs. The latter is in line with previous data from our group that were obtained in experiments in which primary lymphocytes were cultured in the presence of IOH-NPs (Montes-Cobos et al., 2017). Further support comes from in vivo EAE studies where macrophages rather than T and B cells were the main target of IOH-NPs (Montes-Cobos et al., 2017). Given that cells found in solid tissues such as fibroblasts, and less efficiently epithelial cells and myoblasts engulf IOH-NP too, we assume that IOH-NPs are not completely cell type specific for macrophages. This needs to be considered with regard to the efficiency of GC therapy by IOH-NPs in
aGvHD and moreover with respect to their ability to induce potential side-effects, such as myopathy or osteoporosis even though IOH-NP uptake by
osteoblasts has not been tested yet.
Another fundamental aspect of new nanoformulations is that they are being well tolerated, which can be tested for example by tetrazolium-based assays such as MTT (Bahadar et al., 2016). Our results indeed indicate that IOH-NPs, containing Zr are well tolerated by all cell lines. It is noteworthy that data from a recent study indicates that Zr activates the TLR 2, 3, 4 and 9 leading to the release of IL-6, TNFα and IL-1β, which was observed by culturing macrophages in the presence of zirconium particles (Obando-Pereda et al., 2014). Whether
DISCUSSION
121 our IOH-NPs provoke the same reaction in the six cell lines needs to be evaluated in the future and should be tested on an mRNA level or via ELISA.
A common way to study the entry route of nanomedicines is the application of pharmacological inhibitors although such a strategy involves disadvantages, for example that they can manipulate other entry routes and are therefore not entirely specific (Kou et al., 2013, Ivanov, 2008). Furthermore, the effects of the inhibitors can be cell line dependent (Vercauteren et al., 2010). However, pharmacological inhibitors are still attractive as their administration results immediately in blocking of individual pathways although it is suggested to obtain corroborating evidence for example from genetic manipulations of crucial proteins or the application of various inhibitors (Dutta and Donaldson, 2012).
We applied three commonly used inhibitors in well-tolerated concentrations.
CytoD successfully prevented the uptake of IOH-NPs in MH-S cells. This inhibitor caps actin filaments, thereby preventing their assembly and ultimately leading to actin filament disintegration. Actin polymerization is a crucial process for phagocytosis and macropinocytosis, but probably also for all other endocytic pathways though it plays no obligatory role in the formation of clathrin coated vesicles (Fujimoto et al., 2000). Interestingly, MDC had no effect on the uptake capacities of IOH-NPs in MH-S cells, which argues against their internalization via clathrin-mediated endocytosis. MDC is a rather specific inhibitor for clathrin-mediated endocytosis, even though some studies have implemented it in inhibiting macropinocytosis and phagocytosis, too, which however would not be compatible with our observation. Amiloride blocks macropinocytosis when applied at millimolar concentrations and we used it even at mircomolar concentrations (Ivanov, 2008). Indeed, Amiloride prevented the uptake of IOH-NPs in MH-S cells suggesting that internalization occurs mainly via macropinocytosis. Besides that, Amiloride induced cell death to some degree, but it is highly unlikely that the diminished uptake of IOH-NPs can be explained only by this fact. Macropinocytosis is an evolutionary conserved, non-selective form of endocytosis. It mediates the uptake of extracellular fluids and in primary immune cells, such as macrophages and dendritic cells, the uptake of soluble antigens derived from pathogens into relatively large macropinosomes
DISCUSSION
122 (>250 nm), a size which is also suitable for IOH-NPs (Canton, 2018, Palm, 2019). Macrophages have a staggering rate of constant macropinocytosis internalizing their surface every 33 minutes (Steinman et al., 1976). However, the process of macropinocytosis itself is not restricted to primary immune cells, because it also occurs in other cell types such as fibroblasts or epithelial cells (Falcone et al., 2006). The fact that phagocytosis is restricted to professional adepts for example monocytes, macrophages, neutrophils and dendritic cells and in addition that it is initiated by the interaction of the respective receptor on the cellular surface of the particle supports our observed entry route of IOH-NPs (Kuhn et al., 2014). Worth mentioning, our experiments were performed in vitro and nanoparticles can have dynamic entities immediately after administration in vivo, probably unwanted entities, which represent biological obstacles.
Generally, solely about 1 % of injected nanoformulations reach the target tissue, which makes it clear why the path towards clinical licensing is remarkable challenging (Li and Lane, 2019). The rapid removal of nanoformulations from the circulation can be explained by obstacles such as opsonization by plasma proteins, including complement components or antibodies. Subsequently nanoformulations are recognized by the respective receptors on the surface of phagocytes, which lead to their internalization and finally elimination, but most importantly influencing the entry route (Blanco et al., 2015). Whether IOH-NPs undergo opsonization within the blood circulation in vivo and whether this has any impact on the entry route needs to be studied in the future. So far, we observed IOH-NPs are internalized in MH-S cells via macropinocytosis, which occurs most likely via the same way in fibroblasts, epithelial cells and myoblasts in vitro.
Macropinocytosis, more precisely the cargo trapped within the macropinosomes, can have two fates, either the cellular surface, where the cargo is released into the extracellular space, or the lysosomes, which contain enzymes for its degradation (Palm, 2019). Our results indicate that IOH-NPs are indeed travelling to lysosomes where the pharmacological agent betamethasone phosphate is released by hydrolysis of the phosphate-ester bond in the functional anions within the IOH-NPs and becomes immunologically
DISCUSSION
123 active. GCs promote an anti-inflammatory phenotype in macrophages, which involves the downregulation of surface expression markers such as MHCII or CD86, both are crucial for T cell activation (Roszer, 2015). This notion is in agreement with our data, which indicate a dose-dependent reduction of both surface markers in BMDMs when cultured in the presence of BMP-NPs.
Moreover, these results are supported by earlier data from our group. The mRNA expression levels of pro- and anti-inflammatory genes in BMDMs were altered upon co-culturing them with BMP-NPs. As a consequence, Tnfa and Il1b were downregulated, whereas the expression of Cd163 and Ym1 was increased (Montes-Cobos et al., 2017). Recent studies by another group also examined effects of BMP-NPs on MH-S cells in vitro. To provoke an immune response, the cells were treated with LPS and cultured with BMP-NPs for 48 hours.
Subsequently, IL-6 in the supernatant was reduced in a dose-dependent manner (Napp et al., 2018). Similar results were observed when TNFα secretion was analyzed (Heck et al., 2015). Collectively, betamethasone is efficiently released from IOH-NPs within the lysosomes and subsequently exerts immuno-modulatory actions in primary macrophages and MH-S cells. The efficiency of BMP-NPs, compared to the reference drug, is described to be similar or even more efficient (Montes-Cobos et al., 2017).