Introduction of microfluidic mixing technique opens a new door for preparation of the liposomes and lipid-based nanoparticles by on-chip technologies that are applicable in a laboratory and industrial scale. This study demonstrates the role of phospholipid bilayer fragment as the key intermediate in the mechanism of liposome formation by microfluidic mixing in the channel with “herring-bone” geometry used with the instrument NanoAssemblr. The fluidity of the lipid bilayer expressed as fluorescence anisotropy of the probe N,N,N-Trimethyl-4-(6-phenyl-1,3,5-hexatrien-1-yl) was found to be the basic parameter affecting the final size of formed liposomes prepared by microfluidic mixing of an ethanol solution of lipids and water phase. Both saturated and unsaturated lipids together with various content of cholesterol were used for liposome preparation and it was demonstrated, that an increase in fluidity results in a decrease of liposome size as analyzed by DLS. Gadolinium chelating lipids were used to visualize the fine structure of liposomes and bilayer fragments by CryoTEM. Experimental data and theoretical calculations are in good accordance with the theory of lipid disc micelle vesiculation.

Carbon-based nanomaterials (C-BNM) have recently attracted an increased attention as the materials with potential applications in industry and medicine. Bioresistance and proinflammatory potential of C-BNM is the main obstacle for their medicinal application which was documented in vivo and in vitro. However, there are still limited data especially on graphene derivatives such as graphene platelets (GP). In this work, we compared multi-walled carbon nanotubes (MWCNT) and two different types of pristine GP in their potential to activate inflammasome NLRP3 (The nod-like receptor family pyrin domain containing 3) in vitro. Our study is focused on exposure of THP-1/THP1-null cells and peripheral blood monocytes to C-BNM as representative models of canonical and alternative pathways, respectively. Although all nanomaterials were extensively accumulated in the cytoplasm, increasing doses of all C-BNM did not lead to cell death. We observed direct activation of NLRP3 via destabilization of lysosomes and release of cathepsin B into cytoplasm only in the case of MWCNTs. Direct activation of NLRP3 by both GP was statistically insignificant but could be induced by synergic action with muramyl dipeptide (MDP), as a representative molecule of the family of pathogen-associated molecular patterns (PAMPs). This study demonstrates a possible proinflammatory potential of GP and MWCNT acting through NLRP3 activation.

Nanodiamonds (ND), especially fluorescent NDs, represent potentially applicable drug and probe carriers for in vitro/in vivo applications. The main purpose of this study was to relate physical–chemical properties of carboxylated NDs to their intracellular distribution and impact on membranes and cell immunityactivation of inflammasome in the in vitro THP-1 cell line model. Dynamic light scattering, nanoparticle tracking analysis, and microscopic methods were used to characterize ND particles and their intracellular distribution. Fluorescent NDs penetrated the cell membranes by both macropinocytosis and mechanical cutting through cell membranes. We proved accumulation of fluorescent NDs in lysosomes. In this case, lysosomes were destabilized and cathepsin B was released into the cytoplasm and triggered pathways leading to activation of inflammasome NLRP3, as detected in THP-1 cells. Activation of inflammasome by NDs represents an important event that could underlie the described toxicological effects in vivo induced by NDs. According to our knowledge, this is the first in vitro study demonstrating direct activation of inflammasome by NDs. These findings are important for understanding the mechanism(s) of action of ND complexes and explain the ambiguity of the existing toxicological data.

Gadolinium (Gd)-based contrast agents are extensively used for magnetic resonance imaging (MRi). Liposomes are potential nanocarrier-based biocompatible platforms for development of new generations of MRi diagnostics. Liposomes with Gd-complexes (Gd-lip) co-encapsulated with thrombolytic agents can serve both for imaging and treatment of various pathological states including stroke. in this study, we evaluated nanosafety of Gd-lip containing pe-DtpA chelating Gd +3 prepared by lipid film hydration method. We detected no cytotoxicity of Gd-lip in human liver cells including cancer HepG2, progenitor (non-differentiated) HepaRG, and differentiated HepaRG cells. Furthermore, no potential side effects of Gd-lip were found using a complex system including general biomarkers of toxicity, such as induction of early response genes, oxidative, heat shock and endoplasmic reticulum stress, DnA damage responses, induction of xenobiotic metabolizing enzymes, and changes in sphingolipid metabolism in differentiated HepaRG. Moreover, Gd-lip did not show pro-inflammatory effects, as assessed in an assay based on activation of inflammasome NLRP3 in a model of human macrophages, and release of eicosanoids from HepaRG cells. in conclusion, this in vitro study indicates potential in vivo safety of Gd-lip with respect to hepatotoxicity and immunopathology caused by inflammation.Gadolinium (Gd) -based contrast agents are extensively used for magnetic resonance imaging (MRI). Gd +3 forms insoluble phosphate salt in biological fluids and cultivation media. Therefore, Gd +3 is used in complexed forms of soluble chelates. These complexes are believed to be stable and non-toxic because of a high stability constant 1 . Recently, accumulation of Gd-based contrast agents has been demonstrated in various organs like kidney, liver and nervous system. It is supposed that toxic effects can be caused by dissociation of Gd ions from chelated complexes 2,3 . Case reports pointed to the induction of nephrotoxicity, hepatotoxicity and neurotoxicity and rare acute adverse reactions to Gd-based contrast agents were also observed in patients 4 .Liposomes, phospholipid-based vesicles, are widely studied as potential nanocarriers of both MRI contrast agents, including Gd +3 , and drug molecules, such as thrombolytic agents 1,5 . Therefore, liposomes can serve as diagnostic as well as theranostic agents for imaging and treatment of various pathological states and illnesses such as cancer, ischemic stroke and vasculature of different organs including liver and spleen 6,7 . Thus, liposomes represent a favourable platform for a new generation of targeted diagnostic and theranostic systems 8 .Liposomes composed of a Gd-chelating lipid, such as 1,2-distearoyl-sn-glycero-3-p hosphoethanolamine-N-diethylenetriaminepentaacetic acid (PE-DTPA (Gd)), can be easily modified to capture targeting moieties on their surface using different chemistries including copper-free click-chemistry. Moreover, different surface coating of liposomes including polyethylene glycol, hyalu...

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