The interplay between lysosome, protein corona and biological effects of cationic carbon dots: Role of surface charge titratability.

Carbon dots (CDs) are nanoparticles (NPs) with potential applications in the biomedical field. When in contact with biological fluids, most NPs are covered by a protein corona. As well, upon cell entry, most NP are sequestered in the lysosome. However, the interplay between the lysosome, the protein corona and the biological effects of NPs is still poorly understood. In this context, we investigated the role of the lysosome in the toxicological responses evoked by four cationic CDs exhibiting protonatable or non-protonatable amine groups at their surface, and the associated changes in the CD protein corona. The four CDs accumulated in the lysosome and led to lysosomal swelling, loss lysosome integrity, cathepsin B activation, NLRP3 inflammasome activation, and cell death by pyroptosis in a human macrophage model, but with a stronger effect for CDs with titratable amino groups. The protein corona formed around CDs in contact with serum partially dissociated under lysosomal conditions with subsequent protein rearrangement, as assessed by quantitative proteomic analysis. The residual protein corona still contained binding proteins, catalytic proteins, and proteins involved in the proteasome, glycolysis, or PI3k-Akt KEGG pathways, but with again a more pronounced effect for CDs with titratable amino groups. These results demonstrate an interplay between lysosome, protein corona and biological effects of cationic NPs in link with the titratability of NP surface charges.

Surface charge influences protein corona, cell uptake and biological effects of carbon dots.

Carbon dots are emerging nanoparticles (NPs) with tremendous applications, especially in the biomedical field. Herein is reported the first quantitative proteomic analysis of the protein corona formed on CDs with different surface charge properties. Four CDs were synthesized from citric acid and various amine group-containing passivation reagents, resulting in cationic NPs with increasing zeta (ζ)-potential and density of positive charges. After CD contact with serum, we show that protein corona identity is influenced by CD surface charge properties, which in turn impacts CD uptake and viability loss in macrophages. In particular, CDs with high ζ-potential (>+30 mV) and charge density (>2 µmol mg-1) are the most highly internalized, and their cell uptake is strongly correlated with a corona enriched in vitronectin, fibulin, fetuin, adiponectin and alpha-glycoprotein. On the contrary, CDs with a lower ζ-potential (+11 mV) and charge density (0.01 µmol mg-1) are poorly internalized, while having a corona with a very different protein signature characterized by a high abundance of apolipoproteins (APOA1, APOB and APOC), albumin and hemoglobin. These data illustrate how corona characterization may contribute to a better understanding of CD cellular fate and biological effects, and provide useful information for the development of CDs for biomedical applications.

Cationic Carbon Nanoparticles Induce Inflammasome-Dependent Pyroptosis in Macrophages via Lysosomal Dysfunction.

Carbon nanomaterials, including carbon dots (CDs), form a growing family of engineered nanoparticles (NPs) with widespread applications. As the rapid expansion of nanotechnologies raises safety concerns, interaction of NPs with the immune system is receiving a lot of attention. Recent studies have reported that engineered NPs may induce macrophage death by pyroptosis. Therefore, this study investigated whether cationic CDs induce pyroptosis in human macrophages and assessed the role of inflammasome and lysosome in this process. Cationic CDs were synthetized by microwave-assisted pyrolysis of citric acid and high molecular weight branched polyethyleneimine. The NPs evoked a dose-dependent viability loss in THP-1-derived macrophages. A cell leakage, an increase in IL-1ß secretion and an activation of caspase-1 were also observed in response to the NPs. Inhibition of caspase-1 decreased CD-induced cell leakage and IL-1ß secretion, while restoring cell viability. Besides, CDs triggered swelling and loss of integrity of lysosome, and inhibition of the lysosomal enzyme cathepsin B decreased CD-induced IL-1ß secretion. Thus, our data provide evidence that cationic CDs induce inflammasome-dependent pyroptosis in macrophages via lysosomal dysfunction.

A Co-Culture Model of the Human Respiratory Tract to Discriminate the Toxicological Profile of Cationic Nanoparticles According to Their Surface Charge Density.

This study aimed at discriminating with sensitivity the toxicological effects of carbon dots (CDs) with various zeta potential (ζ) and charge density (Qek) in different cellular models of the human respiratory tract. One anionic and three cationic CDs were synthetized as follows: CD-COOH (ζ = -43.3 mV); CD-PEI600 (Qek = 4.70 µmol/mg; ζ = +31.8 mV); CD-PEHA (Qek = 3.30 µmol/mg; ζ = +29.2 mV) and CD-DMEDA (Qek = 0.01 µmol/mg; ζ = +11.1 mV). Epithelial cells (A549) and macrophages (THP-1) were seeded alone or as co-cultures with different A549:THP-1 ratios. The obtained models were characterized, and multiple biological responses evoked by CDs were assessed in the mono-cultures and the best co-culture model. With 14% macrophages, the 2:1 ratio co-culture best mimicked the in vivo conditions and responded to lipopolysaccharides. The anionic CD did not induce any effect in the mono-cultures nor in the co-culture. Among the cationic CDs, the one with the highest charge density (CD-PEI600) induced the most pronounced responses whatever the culture model. The cationic CDs of low charge density (CD-PEHA and CD-DMEDA) evoked similar responses in the mono-cultures, whereas in the co-culture, the three cationic CDs ranked according to their charge density (CD-PEI600 > CD-PEHA > CD-DMEDA), when taking into account their inflammatory effect. Thus, the co-culture system developed in this study appears to be a sensitive model for finely discriminating the toxicological profile of cationic nanoparticles differing by the density of their surface charges.

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

Carbon dots (CDs) are emerging nanomaterial in medicine and pharmacy. To explore the impact of physicochemical characteristics on their safety, we synthesized a library of 35 CDs exhibiting different size, charge, chemical composition and surface coating, using various starting materials (carbon source and passivation reagent) and carbonization procedures. The 35 CDs triggered different levels of viability loss when incubated with human macrophages at 3-200 µg/mL for 24 h. The smaller NPs (10-20 nm) were more toxic that larger ones (40-100 nm), whereas NPs that aggregated in culture medium were more toxic than dispersed ones. A positive correlation was found between CD charge or nitrogen content and toxicity. Furthermore, a greater toxicity was observed for CDs prepared from high molecular weight polyamines, suggesting a role of the CD global density of positive charges, rather than the charge at the CD surface, in the CD toxicity. At last, PEG decoration decreased the toxicity of cationic NPs. In conclusion, the size, aggregation in culture medium, charge, nitrogen content, nature of the passivation agent and synthesis procedure were found to influence CD toxicity, making it difficult to predict CD safety from a single characteristic.