Lysosome mediates toxicological effects of polyethyleneimine-based cationic carbon dots

Abstract

Cationic carbon dots (CDs) have been recently described as nucleic acid carriers with high in vitro and in vivo transfection efficiency and imaging properties. However, developing nanoparticles for biomedical applications requires assessing their safety. In the present study, we characterized the cell uptake and trafficking, as well as the cell viability loss, oxidative stress, inflammation and mitochondrial and lysosomal perturbations evoked by cationic CDs prepared by microwave-assisted pyrolysis of citric acid and high molecular weight branched polyethyleneimine (bPEI25k), using THP-1-derived macrophages. CDs were rapidly internalized by cells and addressed to the lysosomes after their cell entry. The NPs induced a dose-and timedependent loss in cell viability that was associated with oxidative stress and IL-8 release. The CDs triggered also a dose-dependent loss in lysosome integrity, mitochondrial dysfunction and NLRP3 inflammasome activation. Inhibition of the lysosomal protease cathepsin B significantly reduced CD-induced mitochondrial dysfunction and NLRP3 inflammasome activation, suggesting a pivotal role of the lysosome in the toxicological effects of the NPs. Our study provides for the first time a mechanistic pathway for the toxicological effects of bPEI25k-based cationic CDs.

Physicochemical characteristics that affect carbon dot safety: Lessons from a comprehensive study on a nanoparticle library

“…These studies may thus not properly reflect the safety of CDs towards living cells. In agreement with this hypothesis, CDs produced from branched polyethyleneimine 25 kDa (bPEI25k) or obtained from candle soot and subsequently passivated with PEI so they display a net positive charge, were reported to exhibit some cytotoxicity towards cultured cells from various tissue origins (Liu et al, 2012;Ronzani et al, 2018). In contrast, negatively charged CDs were found to have limited toxicity (cell viability loss was less than 20 % at concentration higher than 250 µg/mL) on murine macrophages (Lategan et al, 2018).…”

“…This was even more clearly observed with PEI-based CDs since ranking of these CDs according to EC50 values was as follows: bPEI25k (CD35, EC50=13.7 µg/mL) < bPEI2k (CD30, EC50=23.2 µg/mL) < bPEI600 (CD24, EC50=92.2 µg/mL) ( Figure 5D). After their cell entry, cationic CDs can traffick to the lysosomes (Ronzani et al, 2018;Wu et al, 2017). At the acidic lysosomal pH, difference in number of protonated amino groups should result in difference in the density of positive charges at or close to the CD surface.…”

Density of surface charge is a more predictive factor of the toxicity of cationic carbon nanoparticles than zeta potential

“…Indeed, direct and/or indirect NP interaction and/or damage to cellular organelles such as lysosomes or mitochondria can lead to oxidative stress, which in turn evokes toxicological responses such as inflammation and viability loss. In a previous work, we found that cationic CDs passivated with high molecular weight bPEI evoked a dose–dependent viability loss that was associated with oxidative stress, IL-8 release, mitochondrial perturbation and loss in lysosome integrity in THP-1 cells [ 48 ]. Thus, to get further insight into the link between surface charge/charge density and cytotoxicity of cationic NPs, we investigated these cellular responses in THP-1 and A549 cells exposed to NP1 to NP6, as described in Methods section.…”

“…ζ-potential is widely used to characterize the charge of NPs and to predict NP toxicity, cationic NPs being generally more toxic than anionic ones, due in part to their greater cell uptake and/or their damaging effect on cell and lysosomal membranes [ 25 – 27 , 48 , 63 ]. A correlation was thus found between ζ-potential and hemolytic activity and lung inflammogenicity of polymeric or polystyrene NPs [ 28 , 29 ].…”

“…NP cell uptake was assessed in THP-1 and A549 cells by CLSM and FACS, thanks to the intrinsic fluorescence properties of CDs. CLSM experiments were carried out as previously described [ 48 ]. Briefly, THP-1 and A549 cells were seeded into 8-well IbiTreat µ-Slides at a density of 10 5 and 75.10 3 cells/well, respectively.…”