Natural nanocrystals (sodium urates, calcium oxalates, etc.) or anthropogenic pollutants (nanodiamonds, NDs) of the nanosized range can interact with a plasma membrane (PM) of body cells. The strategy of our organism in the fight against hydrophobic substances is based on their oxidation, conjugation with more dissolved hydrophilic adducts and their subsequent removal. Nanocomposites are promising agents for controlled enhanced permeability of the PM for the delivery of drugs, genes, or fluorescent probes. In addition, nanocrystals are used to detect cell death. Recent data in silico demonstrated the critical effect of nanoparticles size on their ability to interact with PM.
In response to exogenous (bacteria, fungi and tiny nanosized dust), as well as endogenous stimuli in the form of particles (urate crystals and oxalates) granulocyte form the neutrophil extracellular traps (NETs). The process consists of rapid release of chromatin and depends on the active forms of oxygen. Discharged DNA and histones possess antimicrobial and anti-inflammatory factors. Aggregated NETs help to reduce inflammatory response, bind and destroy proinflammatory mediators. Now there is no evidence of the presence of specific receptor-mediated absorption of nanocrystals by neutrophils. However, due to high hydrophobicity, nanocrystals interact directly with the lipid bilayer of the PM. That is why the aim of this study was to investigate the interaction of nanocrystals with a PM of different cell types, particularly immunocompetent, and to determine their effect on development of the immune response. HeLa and Jurkat cell lines were cultured in RPMI media supplemented with fetal calf serum and 5% CO2 at 37°C.
Balb/C mice were used for experiments. Ovalbumin was injected intraperitoneally in the presence or absence of adjuvants at day 1 and day 15 of the experiment. The delayed-type hypersensitivity (DTH) test was carried out 28 d after the first immunization by injection of OVA into the right hind paw. It swelling was assessed by the thickness of the foot 48 h after the injection. For determination of anti-OVA IgG enzyme-linked immunosorbent assay was used. All serum samples were diluted 1:1 000 and incubated at 37 °C. Goat Anti-Mouse IgG-HRP was diluted in washing buffer 1:20 000 and incubated at room temperature. The assay was conducted with 3,3´,5,5´-tetramethybezidine substrate. The reaction was stopped by means of sulfuric acid. The absorbance was read at 450 nm.
The interaction of small NDs with adherent epithelial cells (HeLa) leads to cytotoxicity associated with vacuolization. The same results were observed after adding nanocrystals to the nonadherent cells (Jurkat). Only small NDs (10 nm) induced the damage of PM.
For the analysis of immune response, we used d10, d10PEG, and d1000 nanocrystals as adjuvants in an ovalbumin immunization model. The cellular immune response against ovalbumin was evaluated as DTH reaction. The d1000 and small d10 NDs after PEGylation (d10PEG) showed significantly lower cellular adjuvant activity. It is known that the PEGylation procedure decreases hydrophobicity and only marginally influences the size of the diamonds.
It was shown that the size of nanoparticles plays a crucial role in interaction with lipid bilayer, leading to PM damage. NDs trigger granulocyte activation and NETs formation. In the case of other nuclear cells, the interaction with NDs cause cell death due to loss of membrane integrity. Based on the data of cell interaction with NDs, we proposed this mechanism. The hydrophobic particles of the sizes comparable to the thickness of lipid bilayers (10 nm) penetrate through PM and disturb its barrier function by forming channels around the particles, causing cell swelling and simultaneously activating membrane regeneration by means of endosomes.
The data obtained are of vital importance for understanding of the physiology and biochemical pathways involved in the diseases related to exposure to hydrophobic nanoparticles, like lung disease of workers polishing diamond and mining coal, etc.
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