carbocyanines and Chemical-and-Drug-Induced-Liver-Injury

Because of the low tissue penetration depth and poor photostability of organic cyanine dye, in addition to environmental interference, it is a great challenge to monitor the degree of drug-induced hepatotoxicity by the in vivo detection of peroxynitrite (ONOO-). Herein, we fabricated heptamethine cyanine dye (P-cy7)-coordinated upconversion nanoparticles (UCNPs), namely UCY7, as a fluorescent nanoprobe for evaluating drug-induced hepatotoxicity. Due to the luminescence resonance energy transfer (LRET) between UCNPs and the cyanine dye (P-cy7), the irradiation changed from visible light at 660 nm to near infrared (NIR) light at 980 nm; therefore, the issues of poor photostability and severe photobleaching of cyanine dye can be effectively solved. After injecting via the tail vein, the nanoprobes are rapidly concentrated in the liver. Since the level of ONOO- is up-regulated during the drug-induced liver injury, the LRET between UCNPs and P-cy7 is disrupted to release the upconversion luminescence at 656 nm, while the upconversion luminescence at 800 nm remains constant, thus achieving the ratio-fluorescent imaging (RFLI) of ONOO- in the liver to calibrate the influence of the environment. In addition, the reduction in the absorption of nanoprobes in the presence of ONOO- allows for sensitive photoacoustic imaging (PAI). Based on the RFLI and PAI of the liver, the real-time monitoring and accurate diagnosis of different degrees of hepatotoxicity using the model of Acetaminophen (APAP) induction was achieved successfully, providing a new approach for the clinical evaluation of drug-induced hepatotoxicity.

Topics: Acetaminophen; Animals; Carbocyanines; Chemical and Drug Induced Liver Injury; Female; Fluorescence Resonance Energy Transfer; Fluorescent Dyes; Infrared Rays; Liver; Metal Nanoparticles; Mice; Mice, Inbred BALB C; Peroxynitrous Acid; Photoacoustic Techniques; Up-Regulation

Recent advances in human embryonic and induced pluripotent stem cell-based therapies in animal models of hepatic failure have led to an increased appreciation of the need to translate the proof-of-principle concepts into more practical and feasible protocols for scale up and manufacturing of functional hepatocytes. In this study, we describe a scalable stirred-suspension bioreactor culture of functional hepatocyte-like cells (HLCs) from the human pluripotent stem cells (hPSCs). To promote the initial differentiation of hPSCs in a carrier-free suspension stirred bioreactor into definitive endoderm, we used rapamycin for "priming" phase and activin A for induction. The cells were further differentiated into HLCs in the same system. HLCs were characterized and then purified based on their physiological function, the uptake of DiI-acetylated low-density lipoprotein (LDL) by flow cytometry without genetic manipulation or antibody labeling. The sorted cells were transplanted into the spleens of mice with acute liver injury from carbon tetrachloride. The differentiated HLCs had multiple features of primary hepatocytes, for example, the expression patterns of liver-specific marker genes, albumin secretion, urea production, collagen synthesis, indocyanin green and LDL uptake, glycogen storage, and inducible cytochrome P450 activity. They increased the survival rate, engrafted successfully into the liver, and continued to present hepatic function (i.e., albumin secretion after implantation). This amenable scaling up and outlined enrichment strategy provides a new platform for generating functional HLCs. This integrated approach may facilitate biomedical applications of the hPSC-derived hepatocytes.

Topics: Activins; Albumins; Animals; Biological Transport; Bioreactors; Carbocyanines; Carbon Tetrachloride; Cell Culture Techniques; Cell Differentiation; Cell- and Tissue-Based Therapy; Chemical and Drug Induced Liver Injury; Collagen; Endoderm; Hepatocytes; Lipoproteins, LDL; Liver; Male; Mice; Pluripotent Stem Cells; Sirolimus; Spleen; Transplantation, Heterotopic

Kunming mice (male, weight 20 +/- 2g) were daily intragastric administration of Chinese nutgall extract (0.2 mL/10 g body weight, equal to 8 g Chinese nutgall material/1 kg body weight) for 30 days. Liver tissue mRNA were extracted from normal control group and Chinese nutgall treated group respectively, then were reversely transcribed to cDNA with dUTP labeled by different fluorescence (Cy3, Cy5) as hybridization probes. Both of the cDNA probes were mixed equally in 20 microL of hybridization solution and hybridized with mice complete genome oligonucleotide microarray. The fluorescent signals were acquired by laser scanner and analyzed by GenePix Pro 4.0 software. The biological function analysis and the pathway analysis of differentially expressed genes were performed according to Gene Ontology database. As a result, there were 461 genes differentially expressed in Chinese nutgall treated group, in which 373 genes were function-known and the others were function-unknown. Among the 461 genes, 267 genes were up-regulated and the others were down-regulated. The differentially expressed genes were involved in metabolism, DNA binding and transcription, protein synthesis and modification, cell cytoskeleton and cell adhesion, cell cycle and differentiation, ion channels and transporters, signal transduction, immune response and apoptosis of liver cell. The presented work might be quite important for understanding the pathogenic mechanisms of liver injury induced by Chinese nutgall.

Topics: Animals; Carbocyanines; Chemical and Drug Induced Liver Injury; Drugs, Chinese Herbal; Gene Expression Profiling; In Situ Hybridization; Liver; Male; Mice; Microarray Analysis; Oligonucleotide Array Sequence Analysis; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Signal Transduction