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Our results demonstrate that electrostatic interaction between hemolytic SNPs and model phospholipid membranes is negligible. SNPs induce membrane destabilization and adsorptive processes induced by agglomeration of phospholipid vesicles. The interaction is driven by van der Waals forces at the level of the hydration layer on the vesicles surface.

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The concept of delivering nanoformulations to desired tissues by means of targeting membrane receptors of high local abundance by ligands anchored to the nanocarrier has gained a lot of attention over the last decade. Currently, there is no unanimous opinion on whether surface functionalization of nanocarriers by targeting ligands translates into any real benefit in terms of pharmacokinetics or treatment outcomes. Having examined the published nanocarriers designed to engage with somatostatin receptors, we realized that in the majority of cases targetability claims were not supported by solid evidence of targeting ligand-targeted receptor coupling, which is the very crux of a targetability concept. Here, we present an approach to characterize targetability of mesoporous silica-based nanocarriers functionalized with ligands of somatostatin receptors. The targetability proof in our case comes from a functional assay based on a genetically-encoded cAMP probe, which allows for real-time capture of receptor activation in living cells, triggered by targeting ligands on nanoparticles. We elaborate on the development and validation of the assay, highlighting the power of proper functional tests in the characterization pipeline of targeted nanoformulations.

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Under pathological conditions, the purinergic P2X7 receptor is activated by elevated concentrations of extracellular ATP. Thereby, the receptor forms a slowly dilating pore, allowing cations and, upon prolonged stimulation, large molecules to enter the cell. This process has a strong impact on cell signaling, metabolism, and viability. This study aimed to establish a link between gradual P2X7 activation and pharmacological endpoints including oxidative stress, hydrogen peroxide generation, and cytotoxicity. Mechanisms of cellular stress and cytotoxicity were studied in P2X7-transfected HEK293 cells. We performed real-time monitoring of metabolic and respiratory activity of cells expressing the P2X7-receptor protein using a cytosensor system. Agonistic effects were monitored using exogenously applied ATP or the stable analogue BzATP. Oxidative stress induced by ATP or BzATP in target cells was monitored by hydrogen peroxide release in human mononuclear blood cells. P2X7-receptor activation was studied by patch-clamp experiments using a primary mouse microglia cell line. Stimulation of the P2X7 receptor leads to ion influx, metabolic activation of target cells, and ultimately cytotoxicity. Conversion of the P2X7 receptor from a small cation channel to a large pore occurring under prolonged stimulation can be monitored in real time covering a time frame of milliseconds to hours. Selectivity of the effects can be demonstrated using the selective P2X7-receptor antagonist AZD9056. Our findings established a direct link between P2X7-receptor activation by extracellular ATP or BzATP and cellular events culminating in cytotoxicity. Mechanisms of toxicity include metabolic and oxidative stress, increase in intracellular calcium concentration and disturbance of mitochondrial membrane potential. Mitochondrial toxicity is suggested to be a key event leading to cell death.

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