Effects of Carboxylated Nanodiamonds on Macrophages During and After Differentiation

Nanodiamonds (ND) are a carbon-based nanomaterial that are increasingly being proposed for developing novel imaging techniques, as carriers of biomolecules and therapeutic drugs, as coatings for implants, and for other biomedical applications. The exceptional chemical, mechanical, and optical properties of ND make this material suitable in a wide range of fields. The application of ND in the biomedical field is attractive but requires more in-depth investigation into the safety of ND and its interactions with different cells and systems. The effects of ND on the immune system are not fully understood or investigated and there are several controverting reports regarding ND biocompatibility. Macrophages are found in almost all tissues of the body and are key players in the vertebrate immune system, maintaining homeostasis and initiating immune response to a wide range of pathogens and foreign or host mediators. I hypothesized that ND can affect macrophages and interfere with their functions, and aimed to study interactions of ND with these cells to better understand the potential impact of ND on the immune system. My studies included monitoring both cultured and bone-marrow-derived macrophages in vitro after different exposure conditions and assessment of their effects on cellular processes using molecular laboratory techniques. Results showed that these particles do not significantly increase cell death or changes in cell morphology. These macrophages internalized ND via phagocytic and clathrin-dependent endocytosis in a time- and dose-dependent manner. The internalized ND localized to the cytoplasm without eliciting an inflammatory response in macrophages. Investigations on the macrophage functions showed that treatment of macrophages with ND did not affect their ability to respond to lipopolysaccharides. On the other hand, their endocytic activity was reduced significantly, irrespective of ND dose. Exposure of bone marrow cells to ND early during their differentiation did not affect their morphology or reduce the percent of cells expressing macrophage surface markers. Nonetheless, ND exposure reduced the number of surface markers expressed on each cell. My findings suggest that ND are not cytotoxic to macrophages at the tested concentrations, but they can interfere with macrophage functions and differentiation. Further studies are needed to explore the mechanisms by which ND suppress macrophages endocytic activity and expression of surface markers and the downstream impact of these suppressed immune functions. In addition, the effects of ND on other cells’ immune functions and expression of other immune mediators yet to be studied before concluding the immunotoxicity or compatibility of ND

Interactions of Carboxylated Nanodiamonds With Mouse Macrophages Cell Line and Primary Cells

Nanodiamonds (ND) have attracted significant interest for their use in several biomedical applications. These applications can be very useful if the safety and compatibility of ND are proven. We assessed the effects of ND (100 nm, Carboxylated) on primary macrophages and a macrophage-like cell line and found that these particles are not toxic to these cells at lower concentrations but may interfere with cell functions and differentiation. Internalization of ND by these cells in a time- and dose-dependent manner was mostly via phagocytosis and clathrin-dependent endocytosis and localized to the cytoplasm but not into the nucleus. No significant induction of inflammatory cytokines or reduction in the ability of these cells to respond to lipopolysaccharides (LPS) was noted. However, the endocytic activity of these cells is significantly reduced. In addition, ND exposure reduced the ability of differentiating bone marrow cells to express macrophage surface markers. Measurement of the fluorescence and absorbance of ND-treated cells clearly showed the ability of these particles to produce a signal at different wavelengths. Therefore, it is important to consider interference of ND in different colorimetric and fluorometric assays when testing interactions or effects of ND on cells. Our findings suggest that ND are not cytotoxic to macrophages at the tested concentrations, but it can interfere with macrophage functions and differentiation and may interfere with assays’ result through the production of a signal at different wavelengths

Cyclophilin D Is a Sensor of Nano-Pulse Stimulation

Nano-Pulse Stimulation (NPS), a pulsed power-derived technology, stimulates structural and functional changes in plasma membranes and cellular organelles. NPS induces a Ca2+ influx and opening of the mitochondrial permeability transition pore (mPTP) that dissipates the mitochondrial membrane potential (ΔΨm) and, when sustained, induces regulated cell death. Here we show that in rat cardiomyoblasts (H9C2) cyclophilin D (CypD) is a mitochondrial sensor for NPS as defined by observations that loss of ΔΨm is Ca2+ and mitochondrial reactive oxygen species (mROS) dependent and cyclosporin A (CsA)-sensitive, which are diagnostic qualities for effects on CypD and the mPTP. Mechanistically, NPS stimulates increases in intracellular Ca2+ which enhances mROS in a dose dependent manner. The regulatory role of CypD on mPTP activation, is effectively inhibited at low Ca2+ concentrations and/or by CsA. Although NPS-induced dissipation of ΔΨm is largely Ca2+-dependent, the degree of Ca2+ sensitivities vary among cell types. Nevertheless, knockdown of the proapoptotic protein, APAF-1, and overexpression of the antiapoptotic protein, Bcl-xl, in human Jurkat T lymphocytes (E6.1) did not affect NPS-induced dissipation of ΔΨm or cell death. Taken together, these results indicate NPS induces activation of the mPTP through Ca2+-dependent, mROS-dependent, CsA-sensitive dissipation of the ΔΨm that is independent of caspase activation and insensitive to protection by Bcl-xl.https://digitalcommons.odu.edu/gradposters2021_gradschool/1001/thumbnail.jp

Nanosecond Pulsed Electric Fields (nsPEFs) Modulate Electron Transport in the Plasma Membrane and the Mitochondria

Nanosecond pulsed electric fields (nsPEFs) are a pulsed power technology known for ablating tumors, but they also modulate diverse biological mechanisms. Here we show that nsPEFs regulate trans-plasma membrane electron transport (tPMET) rates in the plasma membrane redox system (PMRS) shown as a reduction of the cell-impermeable, WST-8 tetrazolium dye. At lower charging conditions, nsPEFs enhance, and at higher charging conditions inhibit tPMET in H9c2 non-cancerous cardiac myoblasts and 4T1-luc breast cancer cells. This biphasic nsPEF-induced modulation of tPMET is typical of a hormetic stimulus that is beneficial and stress-adaptive at lower levels and damaging at higher levels. NsPEFs also attenuated mitochondrial electron transport system (ETS) activity (O2 consumption) at Complex I when coupled and uncoupled to oxidative phosphorylation. NsPEFs generated more reactive oxygen species (ROS) in mitochondria (mROS) than in the cytosol (cROS) in non-cancer H9c2 heart cells but more cROS than mROS in 4T1-luc cancer cells. Under lower charging conditions, nsPEFs support glycolysis while under higher charging conditions, nsPEFs inhibit electron transport in the PMRS and the mitochondrial ETS producing ROS, ultimately causing cell death. The impact of nsPEF on ETS presents a new paradigm for considering nsPEF modulation of redox functions, including redox homeostasis and metabolism