Fluorescence lifetime imaging microscopy and time-resolved anisotropy of nanomaterial-induced changes to red blood cell membranes

Abstract

With the use of engineered nano-materials (ENM) becoming more prevalent, it is essential to determine potential human health impacts. Specifically, the effects on biological lipid membranes will be important for determining molecular events that may contribute to both toxicity and suitable biomedical applications. To better understand the mechanisms of ENM-induced hemolysis and membrane permeability, fluorescence lifetime imaging microscopy (FLIM) was performed on human red blood cells (RBC) exposed to titanium dioxide ENM, zinc oxide ENM, or micron-sized crystalline silica. In the FLIM images, changes in the intensity-weighted fluorescence lifetime of the lipophilic fluorescence probe Di-4-ANEPPDHQ were used to identify localized changes to membrane. Time-resolved fluorescence anisotropy and FLIM of RBC treated with methyl-ß-cyclodextrin was performed to aid in interpreting how changes to membrane order influence changes in the fluorescence lifetime of the probe. Treatment of RBC with methyl-ß-cyclodextrin caused an increase in the wobble-in-a-cone angle and shorter fluorescence lifetimes of di-4-ANEPPDHQ. Treatment of RBC with titanium dioxide caused a significant increase in fluorescence lifetime compared to non-treated samples, indicating increased membrane order. Crystalline silica also increased the fluorescence lifetime compared to control levels. In contrast, zinc oxide decreased the fluorescence lifetime, representing decreased membrane order. However, treatment with soluble zinc sulfate resulted in no significant change in fluorescence lifetime, indicating that the decrease in order of the RBC membranes caused by zinc oxide ENM was not due to zinc ions formed during potential dissolution of the nanoparticles. These results give insight into mechanisms for how these three materials might disrupt RBC membranes and membranes of other cells. The results also provide evidence for a direct correlation between the size, interaction-available surface area of the nano-material and cell membrane disruption.

Cholesterol content regulates silica-induced lysosomal membrane permeability

“…We have previously demonstrated that crystalline silica can interact with and disrupt lipid membranes, which resulted in changes to lipid order in the membrane. This is likely through preferential interactions with phosphatidylcholine (DOPC) phospholipid headgroups ( Pavan et al, 2020 ; Sydor et al, 2021 ; Pavan et al, 2022 ). Because of this our current work utilized 100-nm liposomes composed of DOPC with or without cholesterol that were incubated with crystalline silica for the purpose of detecting silica-induced changes to membrane order.…”

“…Silica has been described to interact with lipid membranes and disrupt them, as measured by lipid order changes ( Sydor et al, 2021 ; Pavan et al, 2022 ). Specifically, we have proposed that nearly free silanol groups on the surface of crystalline silica can interact with phosphatidylcholine headgroup lipids and cause a change in lipid order in membranes ( Pavan et al, 2022 ).…”

“…DOPG liposomes in PBS were used to treat cells, while DOPC liposomes in TBS were incubated with silica particles at 37°C with tumbling for 2 h. Lipid order in 100-nm DOPC liposomes was assessed by time-resolved fluorescence anisotropy measurements of the lipophilic probe, Di-4-ANEPPDHQ. The fluorescence probe, Di-4-ANEPPDHQ has been previously demonstrated to be sensitive to changes in the lipid environment surrounding the probe ( Owen et al, 2011 ; Steele et al, 2019 ; Sydor et al, 2020 ; Sydor et al, 2021 ; Pavan et al, 2022 ). Di-4-ANEPPDHQ was dissolved in spectral grade dimethyl sulfoxide (DMSO) and used at a working concentration of 400 nM with the final DMSO concentration being <1%.…”

“…Details of how silica causes LMP remain unclear. However, there are reports suggesting that silica can interact preferentially with phospholipid components of biological membranes and cause changes to the lipid order increasing permeability of those membranes ( Kettiger et al, 2016 ; Wang et al, 2019 ; Sydor et al, 2021 ; Pavan et al, 2022 ). Key surface groups called nearly free silanols (NFS) have been described and are present on the fractured surface of crystalline silica and other types of quartz materials ( Brinker et al, 2020 ; Pavan et al, 2020 ).…”