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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., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023. Published by Elsevier B.V.)

(1) Background: Epiregulin (EREG) is a ligand of EGFR and ErB4 involved in the development and the progression of various cancers including head and neck squamous cell carcinoma (HNSCC). Its overexpression in HNSCC is correlated with short overall survival and progression-free survival but predictive of tumors responding to anti-EGFR therapies. Besides tumor cells, macrophages and cancer-associated fibroblasts shed EREG in the tumor microenvironment to support tumor progression and to promote therapy resistance. Although EREG seems to be an interesting therapeutic target, no study has been conducted so far on the consequences of EREG invalidation regarding the behavior and response of HNSCC to anti-EGFR therapies and, more specifically, to cetuximab (CTX); (2) Methods: EREG was silenced in various HNSCC cell lines. The resulting phenotype (growth, clonogenic survival, apoptosis, metabolism, ferroptosis) was assessed in the absence or presence of CTX. The data were confirmed in patient-derived tumoroids; (3) Results: Here, we show that EREG invalidation sensitizes cells to CTX. This is illustrated by the reduction in cell survival, the alteration of cell metabolism associated with mitochondrial dysfunction and the initiation of ferroptosis characterized by lipid peroxidation, iron accumulation and the loss of GPX4. Combining ferroptosis inducers (RSL3 and metformin) with CTX drastically reduces the survival of HNSCC cells but also HNSCC patient-derived tumoroids; (4) Conclusions: The loss of EREG might be considered in clinical settings as a predictive biomarker for patients that might undergo ferroptosis in response to CTX and that might benefit the most from the combination of ferroptosis inducers and CTX., Competing Interests: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

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.

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-1β 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-1β 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-1β secretion. Thus, our data provide evidence that cationic CDs induce inflammasome-dependent pyroptosis in macrophages via lysosomal dysfunction., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Arezki, Rapp, Lebeau, Ronzani and Pons.)

This study aimed at discriminating with sensitivity the toxicological effects of carbon dots (CDs) with various zeta potential (ζ) and charge density (Q ek ) 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 (Q ek = 4.70 µmol/mg; ζ = +31.8 mV); CD-PEHA (Q ek = 3.30 µmol/mg; ζ = +29.2 mV) and CD-DMEDA (Q ek = 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.

With the growth of nanotechnologies, concerns raised regarding the potential adverse effects of nanoparticles (NPs), especially on the respiratory tract. Adverse outcome pathways (AOP) have become recently the subject of intensive studies in order to get a better understanding of the mechanisms of NP toxicity, and hence hopefully predict the health risks associated with NP exposure. Herein, we propose a putative AOP for the lung toxicity of NPs using emerging nanomaterials called carbon dots (CDs), and in vivo and in vitro experimental approaches. We first investigated the effect of a single administration of CDs on mouse airways. We showed that CDs induce an acute lung inflammation and identified airway macrophages as target cells of CDs. Then, we studied the cellular responses induced by CDs in an in vitro model of macrophages. We observed that CDs are internalized by these cells (molecular initial event) and induce a series of key events, including loss of lysosomal integrity and mitochondrial disruption (organelle responses), as well as oxidative stress, inflammasome activation, inflammatory cytokine upregulation and macrophage death (cellular responses). All these effects triggering lung inflammation as tissular response may lead to acute lung injury.

Background: A positive surface charge has been largely associated with nanoparticle (NP) toxicity. However, by screening a carbon NP library in macrophages, we found that a cationic charge does not systematically translate into toxicity. To get deeper insight into this, we carried out a comprehensive study on 5 cationic carbon NPs (NP2 to NP6) exhibiting a similar zeta (ζ) potential value (from + 20.6 to + 26.9 mV) but displaying an increasing surface charge density (electrokinetic charge, Q ek from 0.23 to 4.39 µmol/g). An anionic and non-cytotoxic NP (NP1, ζ-potential = - 38.5 mV) was used as control., Results: The 5 cationic NPs induced high (NP6 and NP5, Q ek of 2.95 and 4.39 µmol/g, respectively), little (NP3 and NP4, Q ek of 0.78 and 1.35 µmol/g, respectively) or no (NP2, Q ek of 0.23 µmol/g) viability loss in THP-1-derived macrophages exposed for 24 h to escalating NP dose (3 to 200 µg/mL). A similar toxicity trend was observed in airway epithelial cells (A549 and Calu-3), with less viability loss than in THP-1 cells. NP3, NP5 and NP6 were taken up by THP-1 cells at 4 h, whereas NP1, NP2 and NP4 were not. Among the 6 NPs, only NP5 and NP6 with the highest surface charge density induced significant oxidative stress, IL-8 release, mitochondrial dysfunction and loss in lysosomal integrity in THP-1 cells. As well, in mice, NP5 and NP6 only induced airway inflammation. NP5 also increased allergen-induced immune response, airway inflammation and mucus production., Conclusions: Thus, this study clearly reveals that the surface charge density of a cationic carbon NP rather than the absolute value of its ζ-potential is a relevant descriptor of its in vitro and in vivo toxicity.

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., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Objective: S-nitrosogluthatione (GSNO), a S-nitrosothiol, is a commonly used as nitric oxide (NO • ) donor. However, its half-life is too short for a direct therapeutic use. To protect and ensure a sustained release of NO • , the encapsulation of GSNO into nanoparticles may be an interesting option., Methods: In this work, we have investigated the early (4 h) and late (24 h) transcriptomic response of THP-1 human monocytes cells to two doses (1.4 and 6 µM) of either free or Eudragit ® nano-encapsulated GSNO using RNA microarray., Results: After exposure to free GSNO, genes mainly involved in apoptosis, cell differentiation, immune response and metabolic processes were differentially expressed. Although, cells exposed to free or encapsulated GSNO behave differently, activation of genes involved in blood coagulation, immune response and cell cycle was observed in both conditions., Conclusions: These results suggest that the encapsulation of low doses of GSNO into Eudragit ® nanoparticles leads to a progressive release of GSNO making this compound a possible oral therapy for several biomedical applications like inflammatory bowel diseases.

S-Nitrosoglutathione (GSNO) is a good candidate for nitric oxide (NO(•)) delivery, and its nanoformulation improves NO(•) stability and bioavailability. We have compared the effect of empty Eudragit nanoparticles (eENP), GSNO-loaded ENP (gENP), and free GSNO on THP-1 human monocytic cell line. We investigated cellular viability and growth by WST-1 and trypan blue tests. ENP uptake was studied using transmission electron microscopy, confocal microscopy, and flow cytometry. Transcriptomic profiles were obtained using microarray. ENP entered cells by clathrin- and caveolae-mediated endocytosis. Exposure to either free GSNO or gENP induced an activation of genes from the same clusters, in favor of intracellular delivery of GSNO by ENP. GSNO nanoformulation might be a therapeutic option for NO(•) delivery.