Assessment of nanomaterial-induced hepatotoxicity using a 3D human primary multi-cellular microtissue exposed repeatedly over 21 days - the suitability of the in vitro system as an in vivo surrogate.

BACKGROUND: With ever-increasing exposure to engineered nanomaterials (NMs), there is an urgent need to evaluate the probability of consequential adverse effects. The potential for NM translocation to distal organs is a realistic prospect, with the liver being one of the most important target organs. Traditional in vitro or ex vivo hepatic toxicology models are often limiting (i.e. short life-span, reduced metabolic activity, lacking important cell populations, etc.). In this study, we scrutinize a 3D human liver microtissue (MT) model (composed of primary hepatocytes and non-parenchymal cells). This unique experiment benefits from long-term (3 weeks) repeated very low exposure concentrations, as well as incorporation of recovery periods (up to 2 weeks), in an attempt to account for the liver's recovery capacity in vivo. As a means of assessing the toxicological potential of NMs, cell cytotoxicity (cell membrane integrity and aspartate aminotransferase (AST) activity), pro/anti-inflammatory response and hepatic function were investigated. RESULTS: The data showed that 2 weeks of cell culture might be close to limits before subtle ageing effects start to overshadow low sub-lethal NM-induced cellular responses in this test system (adenylate kinase (AK) cytotoxicity assay). We showed that in vitro AST measurement are not suitable in a nanotoxicological context. Moreover, the cytokine analysis (IL6, IL8, IL10 and TNF-α) proved useful in highlighting recovery periods as being sufficient for allowing a reduction in the pro-inflammatory response. Next, low soluble NM-treated MT showed a concentration-dependent penetration of materials deep into the tissue. CONCLUSION: In this study the advantages and pitfalls of the multi-cellular primary liver MT are discussed. Furthermore, we explore a number of important considerations for allowing more meaningful in vitro vs. in vivo comparisons in the field of hepatic nanotoxicology.

Integrated Assessment of Diclofenac Biotransformation, Pharmacokinetics, and Omics-Based Toxicity in a Three-Dimensional Human Liver-Immunocompetent Coculture System.

In vitro hepatocyte culture systems have inherent limitations in capturing known human drug toxicities that arise from complex immune responses. Therefore, we established and characterized a liver immunocompetent coculture model and evaluated diclofenac (DCF) metabolic profiles, in vitro-in vivo clearance correlations, toxicological responses, and acute phase responses using liquid chromatography-tandem mass spectrometry. DCF biotransformation was assessed after 48 hours of culture, and the major phase I and II metabolites were similar to the in vivo DCF metabolism profile in humans. Further characterization of secreted bile acids in the medium revealed that a glycine-conjugated bile acid was a sensitive marker of dose-dependent toxicity in this three-dimensional liver microphysiological system. Protein markers were significantly elevated in the culture medium at high micromolar doses of DCF, which were also observed previously for acute drug-induced toxicity in humans. In this immunocompetent model, lipopolysaccharide treatment evoked an inflammatory response that resulted in a marked increase in the overall number of acute phase proteins. Kupffer cell-mediated cytokine release recapitulated an in vivo proinflammatory response exemplified by a cohort of 11 cytokines that were differentially regulated after lipopolysaccharide induction, including interleukin (IL)-1ß, IL-1Ra, IL-6, IL-8, IP-10, tumor necrosis factor-α, RANTES (regulated on activation normal T cell expressed and secreted), granulocyte colony-stimulating factor, macrophage colony-stimulating factor, macrophage inflammatory protein-1ß, and IL-5. In summary, our findings indicate that three-dimensional liver microphysiological systems may serve as preclinical investigational platforms from the perspective of the discovery of a set of clinically relevant biomarkers including potential reactive metabolites, endogenous bile acids, excreted proteins, and cytokines to predict early drug-induced liver toxicity in humans.

Establishment of a hepatocyte-kupffer cell coculture model for assessment of proinflammatory cytokine effects on metabolizing enzymes and drug transporters.

Elevated levels of proinflammatory cytokines associated with infection and inflammation can modulate cytochrome P450 enzymes, leading to potential disease-drug interactions and altered small-molecule drug disposition. We established a human-derived hepatocyte-Kupffer cell (Hep:KC) coculture model to assess the indirect cytokine impact on hepatocytes through stimulation of KC-mediated cytokine release and compared this model with hepatocytes alone. Characterization of Hep:KC cocultures showed an inflammation response after treatment with lipopolysaccharide and interleukin (IL)-6 (indicated by secretion of various cytokines). Additionally, IL-6 exposure upregulated acute-phase proteins (C-reactive protein, alpha-1-acid glycoprotein, and serum amyloid A2) and downregulated CYP3A4. Compared with hepatocytes alone, Hep:KC cocultures showed enhanced IL-1ß-mediated effects but less impact from both IL-2 and IL-23. Hep:KC cocultures treated with IL-1ß exhibited a higher release of proinflammatory cytokines, an increased upregulation of acute-phase proteins, and a larger extent of metabolic enzyme and transporter suppression. IC50 values for IL-1ß-mediated CYP3A4 suppression were lower in Hep:KC cocultures (98.0-144 pg/ml) compared with hepatocytes alone (IC50 > 5000 pg/ml). Cytochrome suppression was preventable by blocking IL-1ß interaction with IL-1R1 using an antagonist cytokine or an anti-IL-1ß antibody. Unlike IL-1ß, IL-6-mediated effects were comparable between hepatocyte monocultures and Hep:KC cocultures. IL-2 and IL-23 caused a negligible inflammation response and a minimal inhibition of CYP3A4. In both hepatocyte monocultures and Hep:KC cocultures, IL-2RB and IL-23R were undetectable, whereas IL-6R and IL-1R1 levels were higher in Hep:KC cocultures. In summary, compared with hepatocyte monocultures, the Hep:KC coculture system is a more robust in vitro model for studying the impact of proinflammatory cytokines on metabolic enzymes.

Assessment of targeting potential of galactosylated and mannosylated sterically stabilized liposomes to different cell types of mouse liver.

Galactose and Mannose residues were tagged on the surface of n-glutaryl-phosphatidylethanolamine (NGPE) containing liposomes with and without polyethylene glycol of molecular weight 2000 Da conjugated to distearoyl phosphatidylethanolamine (PEG-2000-DSPE). Biodistribution studies showed that sugar bearing liposomes were cleared more rapidly from circulation than those not bearing the sugar moieties. However, the rate of clearance of glycosylated conventional liposomes was much faster than the sugar bearing sterically stabilized liposomes. Intrahepatic distribution studies showed that a substantial amount of conventional liposomes without sugar residues were taken up by both parenchymal (P) (40%) and non-parenchymal (NP) cells (60%). However, incorporation of PEG-2000-DSPE shifted this uptake slightly in favour of parenchymal cells (47%). While ratio of distribution of galactosylated conventional liposomes to P and NP cells was found to be 74:26, galactosylation of sterically stabilized liposomes further enhanced the affinity of these vesicles towards P cells (P:NP ratio being 93:7). Thus, reduced uptake by Kupffer cells was observed with galactosylated sterically stabilized liposomes as compared to conventional liposomes. Whereas, mannosylation of both the liposomes shifted the distribution towards Kupffer cells in an analogous manner. These findings indicate that sterically stabilized liposomes tagged with galactose residues on their surface are more effective in targeting the entrapped material to hepatocytes as compared to conventional liposomes. This approach can therefore be employed for delivering therapeutic agents like drugs, enzymes, genetic materials, anti-sense oligonucleotides selectively to liver P cells for treatment of hepatic disorders.

[An importance of colloid chemical characterization of liposomes for DDS in a large scale production].

Efficacy and safety data of liposomal drugs in a laboratory environment are often not reproduced on an industrial production scale. This is largely due to the fact that the colloid-chemical properties of the liposomes manufactured on a small scale are not reproduced in large scale production. Though the size and the electric charge of liposomes are measured and are adequately specified in relation to the bio-distributions in most developments of liposomes (1), uniformity of lipid components, exposure of bio-chemically important functional groups on the outer surface of liposomes (2), fixed aqueous layer thickness (FALT), number of the lipid bilayers, etc., are dependent upon the scale of production. Nevertheless these properties are not always exactly specified. Uniformity, especially of the functional groups on the membrane surface can be assessed chemically or bio-chemically with fractionated samples, and FALT can be easily determined through electro-chemical means (3). In this review, colloid chemical characterization of liposomes is introduced, FALT as an example, and its importance in a quality control of a liposomal product in an industrial scale production is shown. Methoxy-polyethyleneglycol-diacylglycerol (PEG-DAG) with varying PEG chain length and acyl chains were synthesized, FALT of liposomes coated with PEG-DAG determined and tissue distribution in tumor bearing mice. The higher incorporation ratio of PEG-DAG into liposomal membrane was observed with PEG-DAG with short acyl chains (myristoyl) and a small PEG molecular weight (1000). The easier to incorporate, the easier to be stripped in the serum. The disposition data in the rats well reflected the colloid chemical and in vitro data of the PEG liposomes. Galactosyl-carbonyl-propionyl-polyethyleneglycol-diacylglycerol (Gal-PEG-DAG) with oxyethylene number, n = 10, 20 and 40 were synthesized. The exposure of the galactose residue beyond the fixed aqueous layer of liposomes coated with Gal-PEG-DAG was monitored by a lectin, Ricinus communis agglutinin (RCA) induced agglutination, the half life in the blood after i.v. injection into rats, organ distribution determined and intrahepatic distribution studied. Only the liposomes containing the Gal-PEG10-DAG aggregated with the lectin, indicating that only with this derivative the galactose group was adequately exposed. The Gal-PEG10-DAG liposomes were cleared from the plasma with a half life of 0.3 h. The plasma elimination could be attributed entirely to increased uptake by the liver. The increased liver uptake was almost entirely attributed to increased uptake by the non-parenchymal cell. Incorporation of PEG-DSPE in to the Gal-PEG10-DAG liposomes caused 1) a three-fold increase in blood circulation time, 2) a small but significant decrease in hepatic uptake after 20 h and 3) a significant shift in intrahepatic distribution in favor of the hepatocytes, comparable to that of the control liposomes. In conclusion, therapeutic efficacy and safety of liposomes can be controlled by their colloid chemical, more exactly, surface chemical properties. By setting up reasonable quality control specification of the properties in laboratory and examining the specifications satisfied in upscaling, the efficacy and safety are reproduced in a large scale product.

Pathology of the liver in mucopolysaccharidosis: light and electron microscopic assessment before and after bone marrow transplantation.

Serial liver biopsies were obtained in 20 patients undergoing bone marrow transplantation (BMT) for mucopolysaccharidosis (MPS). The 13 patients with MPS I, one with MPS II, four with MPS III, and two with MPS VI underwent liver biopsy prior to and from 1 to 37 months after BMT. The amount of accumulated glycosaminoglycan (GAG) was assessed by semiquantitation of Kupffer cell staining with colloidal iron and by counting the number of hepatocellular GAG-containing lysosomes in electron micrographs. Eleven of 13 patients with MPS I achieved engraftment, and 10 of the 11 cleared the Kupffer cells and hepatocytes of GAG by 3 to 19 months post-BMT. Two patients with autologous recovery demonstrated persistent hepatocyte inclusions. The three patients with MPS II and MPS VI engrafted and showed clearance of hepatocyte and Kupffer cell GAG by 7 months after BMT. All four patients with MPS III engrafted. Although the Kupffer cells in these patients were cleared of GAG by 12 months after BMT, hepatocellular inclusions persisted in all four. For MPS I, II and VI, donor engraftment was associated with resolution of lysosomal storage material in donor-derived Kupffer cells and untransplanted hepatocytes, indicative of transcellular metabolic correction. Failure of hepatocyte clearance in one case of MPS I and all patients with MPS III suggested a diminished capacity of the graft-derived enzyme to enter the hepatocyte lysosomes in these patients.