AICAR phosphate sale br Experimental br Results and discussi

Experimental
Results and discussion One of the major challenges of oral drug delivery via nanocarriers is the fairly low efficiency of crossing the intestinal barrier. While there is the possibility of a paracellular transport for polar substances below 1000 Dalton [26], the key mechanism of overcoming these natural barriers for the most types of nanomaterials is transcytosis. The limiting factor is often not the uptake into intestinal AICAR phosphate sale but the intracellular fate of the nanomaterial. Specifically, this means a degradation of the nanomaterial via the endolysosomal pathway. In addition, many intracellular pathways and involved proteins in nanoparticle trafficking remain barely researched. Thus, it is essential to understand the molecular mechanisms behind transcytosis of nanomaterials. In this study, we used a combination of apical-to-basal transport in polycarbonate transwells and protein mass spectrometry to identify putative exo- and transcytosis-relevant proteins. To further confirm and extend these results, we were able to elaborately monitor and analyze the intracellular trafficking of nanoparticles in Caco-2 cells expressing eGFP- or mCherry-tagged proteins.
Transcytosis of PS-COOH-NP through Caco-2 cell layers For our experiments, we used carboxyl-functionalized polystyrene nanoparticles (Zeta potential −25 ± 4) with a diameter around 100 nm. Before performing in vitro transcytosis assays, transmission electron microscopy and confocal laser scanning microscopy, synthesized nanoparticles were scanned for possible cytotoxic effects via a quantitative ATP measurement. None of the nanoparticles treatments resulted in a major decrease in metabolic activity regarding concentrations up to 600 µg/mL for 48 h, while the addition of 20% DMSO as a toxic positive control resulted in a decrease to 3% of metabolic activity (Fig. 1A). Similar results were obtained by measuring apoptosis via Annexin V staining (SI Fig. 7), necrosis via propidium iodide staining (SI Fig. 8) and cell proliferation via MTS Assay (SI Fig. 9). After excluding a negative effect of nanoparticles on Caco-2 cells, the development of TEER values before and after nanoparticle addition was determined (SI Fig. 10). After the confirmation that nanoparticles did not have any influence on TEER values, the rate of transcytosis was determined to be around 0.2% of nanoparticle stock solution (Fig. 1B) for nanoparticle concentrations between 75 and 600 µg/mL. This fairly low efficiency is comparable to Caco-2 transcytosis studies performed by other groups using Caco-2 cell systems [8]. In a recent study, Walczak et al. [27] investigated the translocation of polystyrene NPs (50 nm, 100 nm) with different surface charges and chemistry in three in vitro intestinal cell models of increasing complexity (monoculture of Caco-2 cells, co-culture with mucus secreting HT29-MTX cells, tri-culture with M-cells). Beside, charge and surface chemistry dependent effects, they observed, independent from the in vitro model, a size dependent effect (7.8% for 50 nm, 0.8% for 100 nm sized PS-NPs). Overall, these results indicate that our PS-COOH-NP did not have any cytotoxic effects on the cells and were, even though with a very low efficiency, able to successfully transcytose in apical-to-basal direction.
Conclusions Nevertheless, the established methodology kinetically combines the biochemical and visual fingerprint of nanomaterial inside the cell. This could be used to analyze a broader spectrum of nanomaterials as well as transcytosis in multilayer cellular systems and in vivo systems, which will be essential for the development of nanocarriers for oral drug delivery and for the risk assessment of nanoplastics.
Introduction The cortical actin network plays crucial roles in a number tasks that are essential for cell survival, growth and communication. It comprises of a dense layer of actomyosin fibres adjacent to the plasma membrane which is crucial in regulating the access of secretory vesicles to their docking and fusion sites in neurosecretory cells (Trifaro et al., 1992, Trifaro et al., 2008). Considerable efforts have been undertaken to decipher the function of the cortical actin network and supporting proteins such as myosins (Berg et al., 2001, Papadopulos et al., 2013a, Papadopulos et al., 2013b), adhesion molecules and small GTPases (Gasman et al., 2003, Hall and Nobes, 2000) as well as lipid modulators (Tanguy et al., 2016) in regulated exocytosis. The close proximity of the cortical actin network to the plasma membrane implicates an intricate interplay between actomyosin and both exo- and endocytic membrane structures. Indeed, the cortical actin network has distinct abilities to affect the outcome and dynamics of exo- and endocytosis (Gutiérrez, 2012, Meunier and Gutiérrez, 2016).