The human HepaRG™ cell line is the closest surrogate to primary culture of hepatocytes (PHH) for toxicology studies. However, differentiated HepaRG™ cells express low levels of the cytochrome P450 2D6 (CYP2D6) involved in the biotransformation of many drugs. Herein, progenitor HepaRG™ cells were transduced using lentiviral particles encoding both human CYP2D6 and GFP proteins. The resulting transgenic HepaRG™ cells stably expressed catalytically active CYP2D6 at levels close to those observed in PHH from rapid metabolizers and HepaSH™ hepatocytes. In CYP2D6 transgenic HepaRG™ cells, tramadol was metabolized into both N- and O-desmethyl tramadol as seen in humans while parental HepaRG™ cells produced only N-desmethyl tramadol. Following treatment with perhexiline, the CYP2D6 expressing HepaRG™ cells exhibited higher IC50 values and reduced mitochondrial damages compared to those found in parental cells. Transcriptomic analysis revealed that the expression of CYP2D6 did not significantly affect the cells’ ability to proliferate and differentiate or compromise key hepatocyte-specific functions. However, we identified a small number of genes, including NXF3 and TRIM63, which were up-regulated in transgenic cells. Using CRISPR/Cas9-mediated knockdown of GFP and/or CYP2D6 sequences, we demonstrated that NXF3 mRNA and protein inductions were triggered by the lentiviral mRNA encoding GFP and CYP2D6 rather than by genomic transgene integration. Together, these findings establish CYP2D6-transgenic HepaRG™ cells as an optimized and reliable hepatocyte-like model for studying the metabolism and toxicity of CYP2D6 substrates. Our results also support the hypothesis that the NXF3 gene may be a marker of cellular response to the expression of a lentiviral chimeric mRNA.

Pesticides are increasingly recognized to be hepatotoxic, but less is known about their toxicity in metabolic dysfunction-associated steatotic liver disease (MASLD). Herein, differentiated HepaRG cells cultured for 2 weeks without (-FA) or with (+FA) a mixture of fatty acids were treated with different pesticides, including maneb and mancozeb, during the same period. While maneb and mancozeb did not induce neutral lipid accumulation in -FA-HepaRG cells, they worsened steatosis in +FA-HepaRG cells. MnCl2 treatment reproduced these effects. Maneb or MnCl2 impaired very low-density lipoprotein (VLDL) secretion and increased fatty acid uptake. Zinc supplementation restored VLDL secretion, reduced fatty acid uptake, and prevented steatosis worsening in +FA-HepaRG cells treated with mancozeb or MnCl2. Maneb, or MnCl2, also reduced the mRNA expression and activity of several cytochromes P450 in +FA- and -FA-HepaRG cells. This was associated with impaired biotransformation of diazinon. These findings could have major pathophysiological consequences in dithiocarbamate-exposed individuals with MASLD.

The lack of understanding of polyplexes stability and their dissociation mechanisms, allowing the release of DNA, is currently a major limitation in non-viral gene delivery. One proposed mechanism for DNA-based polyplexes dissociation is based on the electrostatic interactions between polycations and biological polyanions, such as glycosaminoglycans (GAGs). This work aimed at investigating whether GAGs such as heparin, chondroitin sulphate and hyaluronic acid promote the dissociation of PEI/DNA polyplexes. We studied the electrostatic complexation between branched poly(ethyleneimine) (b-PEI25) and polyanions (model DNA and GAGs) through conductivity and ζ-potential measurements. The formation of b-PEI25/polyanion polyplexes through electrostatic interactions was analyzed in depth, providing key insights into charge stoichiometry, morphology, thermodynamics and physicochemical characteristics. The stability of polyplexes was tested in the presence of the different GAGs. Heparin was found to be the only polyanion capable of releasing peGFP-C3 plasmid from polyplexes, complexing stoichiometrically with the free b-PEI25 in excess, before releasing the plasmid. The ability of GAGs to disrupt polyplexes and release DNA was correlated with the thermodynamic characteristics of b-PEI25/polyanions complexation. Our findings indicate that heparin's strong interaction with PEI and its high charge density, compared to other GAGs and polyanions, are pivotal in determining complex stability and promoting DNA release.

The liver's regenerative capacity is underscored by the plasticity potential of adult hepatocytes. In this context, hepatocyte-to-cholangiocyte transdifferentiation (HCT) has been ascribed with pro-regenerative functions in animal models and is a feature of end-stage human chronic liver diseases. While dampened activities of hepatocyte identity transcription factors (TFs) underlay HCT, how the cholangiocyte transcriptional program is implemented is poorly defined. Here, we identify that HCT does not involve transitioning through a hepatoblast-like transcriptional program. Furthermore, we show that HCT primarily involves induction of the archetypal transcriptional program of monopolarized epithelial cells initially repressed in hepatocytes. Indeed, HCT requires relieving H3K27me3-mediated and polycomb-dependent epigenetic silencing of epithelial TF encoding genes including Grainyhead Like Transcription Factor 2 (GRHL2). Ectopic expression of GRHL2 in hepatocytes, including in vivo in the adult mouse liver, induces epithelial genes reminiscent of those activated during HCT. Finally, GRHL2 is detected in human hepatocytes undergoing HCT as evidenced using samples from end-stage chronic liver diseases. Hence, HCT is a process chiefly characterized by induction of a conventional epithelial transcriptional program originally lacking in hepatocytes promoted by derepression of the master epithelial TF GRHL2.

Using the phage display technology, we identified a novel peptide, P11Chol, which preferentially binds to both human and rat cholangiocytes. Peptide P11Chol alignment with protein databases evidenced strong similarities with a highly conserved peptide motif from BamA/TamA-like outer membrane proteins expressed in enterobacteriaceae belonging to Pseudomonadota phylum including Photorhabdus , Providencia , Acinetobacter , Salmonella enterica and Helicobacter pylori species. In addition, we showed that Providencia stuartii bacteria were able to bind to cholangiocytes-like HepaRG cells in vitro and that P11Chol modulated this interaction suggesting the possible involvement of BamA/TamA-like outer membrane proteins in cell adhesion and/or internalization of Providencia stuartii bacteria. Using fluorescent P11Chol peptide, we next developed a flow cytometry procedure to detect and isolate rat and human liver epithelial cells from hepatic cell suspension obtained after collagenase dissociation of liver parenchyma. Three distinct P11Chol-positive rat liver epithelial cell lines (RLEC) were established, which produced functional cholangiocytes capable to form cyst-like structures in vitro and to maintain expression of specific functions in hepatocytes in coculture. The characterization of these three RLEC lines evidenced functional differences that support the concept of small and large cholangiocytes exhibiting different functional phenotypes within the intrahepatic bile tree. ### Competing Interest Statement The authors have declared no competing interest. Lebanese Association for Scientific Research (LASeR, Lebanon) Institut National de la Santé et de la Recherche Médicale (Inserm, France) Rennes Métropôle (France) Université de Bretagne Loire (UBL) Convention Industrielle de Formation par la Recherche, CIFRE n°221207A10 Convention Industrielle de Formation par la Recherche, CIFRE n°221206A10, Ministère de l’Enseignement Supérieur et de la Recherche, https://ror.org/03sjk9a61

Natural and synthetic polycations are commonly used in DNA- and RNA-based transfection for their ability to condense and protect nucleic acids, and the subsequent cellular uptake. While the formation of these complexes is well-documented, fewer studies have examined their stability under both extracellular and intracellular conditions. Chitosan, a biodegradable natural polysaccharide, has shown promise for gene delivery. However, its pKa of similar to 6.5 makes its ionization state highly sensitive to pH fluctuations around this value. In this study, we examined how pH-induced changes influence the stability of chitosan/peGFP-C3 complexes and their effects on cell viability, proliferation, and transfection efficiency in HEK293T cells. The highest transfection efficiency was achieved by transfecting the complexes at pH 6.5, followed by a medium replacement at pH 7.1, particularly with high molecular weight chitosans. Cell viability was found to be higher at pH 7.1, whereas acidic conditions (pH 6.5) affected proliferation, leading to S-phase accumulation, suggesting potential interference with cell cycle progression. These findings underscore the critical role of pH in optimizing gene delivery and maintaining favorable cell growth conditions.

Primary cultures of Human Hepatocytes (PHH) are the gold standard to investigate drug-hepatotoxicity in vitro, however, large-scale studies using these primary liver cells are not possible because of the shortage in liver biopsies. HepaRG model is often considered as the closest surrogate to PHH for toxicity studies in vitro. However, differentiated HepaRG cells express very low levels of the cytochrome P450 2D6 (CYP2D6) protein, which is essential for the biotransformation of nearly 25% of drugs on the market. To overcome this limitation, infection of progenitor HepaRG cells were performed using lentiviral particles containing a transgene encoding a single mRNA translated into a polypeptide undergoing proteolytic cleavage via the T2A peptide to produce both CYP2D6 and GFP. Differentiated HepaRG cells transduced with lentivirus stably expressed GFP and catalytically active human CYP2D6 enzyme at levels close to those found in high PHH metabolizers. As expected, CYP2D6 protein was found mostly located in the endoplasmic reticulum. Using the CYP2D6 transgenic HepaRG cells, we showed that tramadol was metabolized in both, N- and O-desmethyl tramadol as observed in human serum in contrast with the production of N-desmethyl tramadol only in parental HepaRG cells via the CYP3A4 catalytic activity. Similarly, after perhexiline (PHX) treatments, higher IC50 were found in CYP2D6 expressing HepaRG cells associated to lower mitochondrial damages compared to those found in parental cells for the same PHX concentrations. Gene profiling between parental and transgenic cells demonstrated that the CYP2D6 expressing HepaRG cells had kept their ability to proliferate and differentiate with low impact on the expression of the hepatocyte specific functions. However, we identified a limited set of genes such as NXF3 and TRIM63, which were up-regulated by the mRNA encoded by the lentiviral transgene. Together, these data confirmed that the CYP2D6 transgenic HepaRG cells represent a suitable optimized transgenic model of HepaRG cells to evaluate biotransformation and toxicity of specific compounds metabolized by CYP2D6. ### Competing Interest Statement The authors have declared no competing interest.

The development of natural or synthetic polycations able to interact with nucleic acids and condense them into nanoparticles known as polyplexes, faces several unresolved challenges at the cellular level. Key issues include the intracellular trafficking of polyplexes, the endosomal escape and the release of nucleic acids into the cytosol, which are considered major bottlenecks for efficient protein expression. Here, we aim at gaining fundamental insights into the stability of polyplexes in biological media and their uptake and intracellular trafficking, while correlating data of the expression of reporter protein with both the molecular characteristics of various poly(ethylenimines) (PEI) and the physicochemical characteristics of PEI/peGFP-C3 polyplexes. For this, we chosen four samples of PEI, selected as a model polycation, with different molecular weights (Mw = 0.8, 20, 25 and 60 kg/mol) and structures (linear and branched). We found that the in vitro and in vivo stability of PEI/peGFP-C3 polyplexes, their cell internalization and transfection efficiency is dependent on the variation of polycation Mw and structure, as well as the intrinsic properties of polyplexes, such as the charge ratio (R=[N+]/[P-]). A relation between the percentage of positive cells to green fluorescent protein (GFP) and the amount of internalized nucleic acid (cyanine 5-peGFP-C3) allowed revealing the molecular characteristics of PEI promoting both higher both cell internalization and GFP expression on HEK293T cells. In the long term, the outcome of this work will be to propose guidelines to help design more effective, and less cytotoxic non-viral gene carriers with a great potential for new therapeutic applications. ### Competing Interest Statement The authors have declared no competing interest.

Lipid nanocapsules (LNCs) used as nanomedicine have been developed to enhance pharmacokinetics and decrease side effects of drugs, particularly for cancer therapies. After intravenous administration, LNCs possess an important hepatic tropism however, few data exist about their toxicity and even less after repeated exposure. This study aimed to assess the in vitro toxicity and internalization of unloaded LNCs, of 50 and 100 nm size, on HepG2 and HepaRG liver cell lines. Internalization of the 50 nm LNCs was slower compared to the 100 nm LNCs and both LNCs exhibited a higher toxicity on cancerous HepG2 cells compared to differentiated HepaRG cells. LNCs were mainly internalized via caveolin-mediated endocytosis in both cell lines. Upon chronic exposure, the toxicity of LNCs on HepaRG cells increased, although the pathways of internalization remained unchanged. Cell death studies have demonstrated an involvement of ferroptosis, but not of apoptosis. After acute and repeated exposures on HepaRG cells, the 100 nm LNCs showed a good safety profile. Finally, LNCs induced a more significant toxicity associated with faster internalization in the HepG2 cancerous model than in the differentiated HepaRG cells. This provides good evidence for LNCs to potentialize the cytotoxic effects of an active drug on liver cancer cells.

Polysaccharide-based nanogels offer a wide range of chemical compositions and are of great interest due to their biodegradability, biocompatibility, non-toxicity, and their ability to display pH, temperature, or enzymatic response. In this work, we synthesized monodisperse and tunable pH-sensitive nanogels by crosslinking, through reductive amination, chitosan and partially oxidized maltodextrins, by keeping the concentration of chitosan close to its overlap concentration, i.e. in the dilute and semi-dilute regime. The chitosan/maltodextrin nanogels presented sizes ranging from 63 +/- 9 to 279 +/- 16 nm, showed quasi-spherical and cauliflower-like morphology, reached a -potential of +36 +/- 2 mV and maintained a colloidal stability for up to 7 weeks. It was found that the size and surface charge of nanogels depended both on the oxidation degree of maltodextrins and chitosan concentration, as well as on its degree of acetylation and protonation, the latter tuned by pH. The pHresponsiveness of the nanogels was evidenced by an increased size, owed to swelling, and -potential when pH was lowered. Finally, maltodextrin-chitosan biocompatible nanogels were assessed by cell viability assay performed using the HEK293T cell line.

In the past years, we have designed biodegradable poly(benzyl malate) (PMLABe73) homopolymer and amphiphilic poly(ethylene glycol)-b-PMLABe (PEG42-b-PMLABe73) copolymer and several modified (co)polymers to produce biocompatible polymeric nanoparticles (NPs) capable of targeting hepatic cells in vitro with the goal to develop applications in the treatment of liver diseases. The current study aimed at comparing the uptake of PMLABe73 PEG42-b-PMLABe73-based NPs in human hepatic HepaRG cells, primary macrophages and peripheral blood mononuclear cells (PBMC). The uptake of NPs prepared from PEG42-b-PMLABe73 was significantly lower than that of PMLABe73 in both hepatic cells and macrophages. In addition, the NPs uptake by HepaRG cells was inversely correlated to the density of PEG present on their surface. In contrast, the internalization of with PMLABe-based NPs by human macrophages was not affected by low PEG densities, only uptake of fully pegylated PEG42-b-PMLABe73based-NPs was significantly decreased. Herein, we also showed that PMLABe-based NPs did not strongly accumulated in PBMC, T lymphocytes and neutrophils while monocytes showed slightly higher uptake of these NPs. Moreover, we further demonstrated that PMLABe-derived NPs by did not trigger inflammasome activation and secretion of pro-inflammatory cytokines neither in macrophages nor HepaRG cells. Then, we demonstrated that peptide GBVA10-9 derived from George Baker (GB) Virus A, known to exhibit a good hepatotropism did not significantly affect the uptake of PMLABe73-based NPs in HepaRG cells and macrophages, when grafted onto these NPs. The present results demonstrate that PMLABe-derived NPs are very efficiently internalized in both macrophages and hepatocytes but not in PBMC and reinforce our previous reports regarding their biocompatibility. ### Competing Interest Statement The authors have declared no competing interest.

The worldwide and intensive use of phytosanitary compounds results in environmental and food contamination by chemical residues. Human exposure to multiple pesticide residues is a major health issue. Considering that the liver is not only the main organ for metabolizing pesticides but also a major target of toxicities induced by xenobiotics, we studied the effects of a mixture of 7 pesticides (chlorpyrifos-ethyl, dimethoate, diazinon, iprodione, imazalil, maneb, mancozeb) often detected in food samples. Effects of the mixture was investigated using metabolically competent HepaRG cells and human hepatocytes in primary culture. We report the strong cytotoxicity of the pesticide mixture towards hepatocytes-like HepaRG cells and human hepatocytes upon acute and chronic exposures at low concentrations extrapolated from the Acceptable Daily Intake (ADI) of each compound. Unexpectedly, we demonstrated that the manganese (Mn)-containing dithiocarbamates (DTCs) maneb and mancozeb were solely responsible for the cytotoxicity induced by the mixture. The mechanism of cell death involved the induction of oxidative stress, which led to cell death by intrinsic apoptosis involving caspases 3 and 9. Importantly, this cytotoxic effect was found only in cells metabolizing these pesticides. Herein, we unveil a novel mechanism of toxicity of the Mn-containing DTCs maneb and mancozeb through their metabolization in hepatocytes generating the main metabolite ethylene thiourea (ETU) and the release of Mn leading to intracellular Mn overload and depletion in zinc (Zn). Alteration of the Mn and Zn homeostasis provokes the oxidative stress and the induction of apoptosis, which can be prevented by Zn supplementation. Our data demonstrate the hepatotoxicity of Mn-containing fungicides at very low doses and unveil their adverse effect in disrupting Mn and Zn homeostasis and triggering oxidative stress in human hepatocytes.

Tamoxifen, the gold standard drug for endocrine therapy for breast cancer, modulates the phosphorylation status of the TAU protein in Alzheimer's disease by inhibiting CDK5 kinase activity. Its binding to p25 prevents CDK5/p25 complexation and hence a decrease of CDK5 activity. In breast tumors, this complex is involved in the proliferation and survival of cancer cells, as well as in the disease's prognosis. Still, the molecular stability of the CDK5/p25 complex following tamoxifen exposure in this cancer type has not yet been clearly deciphered. Here, we report the functional characterization of CDK5 and its p25 regulatory subunit in the absence and presence of tamoxifen. In addition, two novel inhibitors of the kinase activity of the CDK5/p25 complex are identified, both of which would reduce the risk of recurrence of estrogen receptor-positive (ER+) breast cancers and prevent drawbacks induced by tamoxifen exposure. Accordingly, 6His-CDK5 and 6His-p25 have been expressed and purified. Fluorescence anisotropy measurements have been used to assess that the two proteins do form an active complex, and thermodynamic parameters of their interaction were measured. It was also confirmed that tamoxifen directly binds to p25 and inhibits CDK5 kinase activity. Similar observations were obtained using 4-hydroxytamoxifen, an active metabolized form of tamoxifen. Two novel compounds have been identified here that harbor a benzofuran moiety and were shown to target directly p25, and their bindings resulted in decreased CDK5 kinase activity. This encouraging alternative opens the way to the ensuing chemical optimization of this scaffold. It also promises a more specific therapeutic approach that may both tackle the pathological signaling in breast cancer and provide a potential new drug for Alzheimer's disease.

The goal of this study was to establish a procedure for gene delivery mediated by cationic liposomes in quiescent differentiated HepaRG™ human hepatoma cells. We first identified several cationic lipids promoting efficient gene transfer with low toxicity in actively dividing HepG2, HuH7, BC2 and progenitor HepaRG™ human hepatoma cells. The lipophosphoramidate Syn1-based nanovector, which allowed the highest transfection efficiencies of progenitor HepaRG™ cells, was next used to transfect differentiated HepaRG™ cells. Lipofection of these cells using Syn1-based liposome was poorly efficient most likely because the differentiated HepaRG™ cells are highly quiescent. Thus, we engineered the differentiated HepaRG™ Mitogenic medium supplement (ADD1001) that triggered robust proliferation of differentiated cells. Importantly, we characterized the phenotypical changes occurring during proliferation of differentiated HepaRG™ cells and demonstrated that mitogenic stimulation induced a partial and transient decrease in the expression levels of some liver specific functions followed by a fast recovery of the full differentiation status upon removal of the mitogens. Taking advantage of the proliferation of HepaRG™ cells, we defined lipofection conditions using Syn1-based liposomes allowing transient expression of the cytochrome P450 2D6, a phase I enzyme poorly expressed in HepaRG cells, which opens new means for drug metabolism studies in HepaRG™ cells.

In order to identify the peptides, selected from the literature, that exhibit the strongest tropism towards human hepatoma cells, cell uptake assays were performed using biotinylated synthetic peptides bound to fluorescent streptavidin or engrafted onto nanoparticles (NPs), prepared from biotin-poly(ethylene glycol)-block-poly(benzyl malate) (Biot-PEG-b-PMLABe) via streptavidin bridging. Two peptides, derived from the circumsporozoite protein of Plasmodium berghei- (CPB) and George Baker (GB) Virus A (GBVA10-9), strongly enhanced the endocytosis of both streptavidin conjugates and NPs in hepatoma cells, compared to primary human hepatocytes and non-hepatic cells. Unexpectedly, the uptake of CPB- and GBVA10-9 functionalized PEG-b-PMLABe-based NPs by hepatoma cells involved, at least in part, the peptide binding to apolipoproteins, which would promote NP’s interactions with cell membrane receptors of HDL particles. In addition, CPB and GBVA10-9 peptide–streptavidin conjugates favored the uptake by hepatoma cells over that of the human macrophages, known to strongly internalize nanoparticles by phagocytosis. These two peptides are promising candidate ligands for targeting hepatocellular carcinomas.

We recently demonstrated the strong tropism of George Baker (GB) Virus A (GBVA10-9) and Plasmodium circumsporozoite protein (CPB) derived synthetic peptides towards hepatoma cells. In a first approach, these peptides were covalently bound to poly(benzyl malate) (PMLABe73) and poly(ethylene glycol)-block-PMLABe73 (PEG62-b-PMLABe73) (co)polymers, and corresponding peptide-decorated nanoparticles (NPs) were prepared by nanoprecipitation. We showed that peptide enhanced NPs internalization by hepatoma cells. In the present work, we set up a second strategy to functionalize NPs prepared from PMLABe73 derivates. First, maleimide-functionalized PMLABe73 (Mal-PMLABe73) and PEG62-b-PMLABe73 (Mal-PEG62-b-PMLABe73) were synthesized and corresponding NPs were prepared by nanoprecipitation. Then, peptides (GBVA10-9, CPB and their scramble controls GBVA10-9scr and CPBscr) with a thiol group were engrafted onto the NPs’ maleimide groups using the Michael addition to obtain peptide functionalized NPs by post-formulation procedure. These peptide-modified NPs varied in diameter and dispersity depending on the considered peptides and/or (co)polymers but kept their spherical shape. The peptide-functionalized NPs were more efficiently internalized by HepaRG hepatoma cells than native and maleimide-NPs with various levels relying on the peptide’s nature and the presence of PEG. We also observed important differences in internalization of NPs functionalized by the maleimide-thiol-peptide reaction compared to that of NPs prepared from peptide-functionalized PMLABe73 derivatives.