Bioavailability of NanoCurc™ in liver tissue. (a) Mice (n = 3 in each group) were treated with single intraperitoneal (i.p.) 25 mg/kg dose of NanoCurc™ (NC), void nanoparticles (Poly) or an equivalent oral gavage of 25 mg/kg of free curcumin (FC). Curcumin bioavailability was determined by high-performance liquid chromatography (HPLC) in liver tissue obtained 12 h after treatment. (b) Mice (n = 3 in each group) were treated with three i.p. 25 mg/kg doses equally spaced over 24 h of NanoCurc™, Poly and phosphate-buffered saline (PBS). Curcumin bioavailability in whole liver tissue was determined by HPLC 6 h after the last dose. (c) Mice (n = 3 in each group) were treated with three i.p. 25 mg/kg doses equally spaced over 24 h of NC, poly and PBS. Hepatocytes (HEPs) and non-parenchymal cells (NPCs) were isolated from the liver. Curcumin bioavailability in HEPs and NPCs was determined by HPLC 6 h after the last dose. Data were expressed as mean ± s.e.m.,*P < 0.01, **P < 0.001, t-test, from two experiments. ^§§indicates below limit of detection.

NanoCurc™ inhibits carbon tetrachloride (CCl₄)-induced liver injury and fibrosis. Mice were treated with intraperitoneal control (a, c) or NanoCurc™ (b, d) injection and CCl₄ according to the treatment protocol in Materials and Methods. (a, b) Methacarn-fixed liver tissues were stained with Sirius Red, which highlights collagen deposition in dark red. Representative photomicrographs (× 10 objective). (c, d) The extent of fibrosis was quantitatively determined using Threshold Image Analysis to highlight fibrosis (orange color) in 10 randomly selected photomicrographs (× 10 objective) after conversion to 8-bit gray scale. (e) The percentage of fibrosis calculated as the area of fibrosis divided by the total image area multiplied by 100. (f) Hydroxyproline assay was carried out to quantify the collagen content. Data were expressed as mean ± s.e.m.,*P < 0.05, **P < 0.001, t-test, from two experiments.

NanoCurc™ attenuates genes associated with stellate cell activation and fibrosis while stimulating pro-apoptotic gene expression. Liver tissue harvested from untreated mice (PBS), void polymer (Poly) or NanoCurc™ (NC) injections along with carbon tetrachloride (CCl₄) according to the treatment protocol in Materials and Methods. Gene expression levels for transcripts encoding for Collagen A (COL A), fibronectin 1 (FN-1), transforming growth factor-β (TGFB) and peroxisome proliferator-activated receptor gamma (PPARG) were determined after isolation of RNA with real-time reverse transcription-polymerase chain reaction (RT-PCR). Gene expression was normalized with hprt and fold expression was determined using the ΔΔC_t method in which untreated liver tissue was set to relative expression level of 1.0. Data were expressed as mean ± s.e.m., **P < 0.001, in comparing Ctrl vs NC, t-test; control n = 4, NC n = 5, representative of two experiments.

NanoCurc™ prevents carbon tetrachloride (CCl₄) associated liver injury and inflammation. The mice were injected with either phosphate-buffered saline (PBS), void nanoparticles (Poly) or NanoCurc™ (NC) injections along with CCl₄ according to the treatment protocol in Materials and Methods. (a) Liver damage was determined by measuring serum alanine aminotransferase (ALT) activity (expressed in Sigma Frankel units) and (b) intrahepatic tumor necrosis factor (TNF)-α was determined by enzyme-linked immunosorbent assay (ELISA). (c and d) Relative interleukin (IL)-6 and TNF-α mRNA expression was determined with real-time reverse transcription-polymerase chain reaction (RT-PCR) using the ΔΔC_t method with histone 2AZ and hprt serving as a reference genes. PBS-negative control liver tissue was set to relative expression level of 1.0. Data were expressed as mean ± s.e.m., *P < 0.05, **P < 0.001, t-test, from two experiments.

NanoCurc™ increases redox capacity of the hepatocytes. (a) Free glutathione (GSH), (b) oxidized GSH (c), total GSH and (d) the ratio of free GSH to oxidized glutathione disulfide (GSSG) were determined as described in Materials and Methods in liver lysates harvested 48 h after CCl₄ treatment in void polymer- (Poly) and NanoCurc™- (NC) treated mice. Data were expressed as mean ± s.e.m., *P values as indicated, t-test, Poly n = 4, NC n = 5.

In vitro NanoCurc™ treatment induces stellate cell apoptosis. Immortalized rat stellate cells, hepatic stellate cell line (HSC-T6), were plated and treated with the indicated doses of free curcumin (FC) dissolved in 0.1% dimethylsulfoxide (DMSO) or an equivalent dose of NanoCurc™ (NC) in plating media. Stellate cells were treated with 20, 40 and 80 µM of FC or NC. After treatment, cells were fixed with 3.7% paraformaldehyde, stained with 4′,6-diamidino-2-phenylindole (DAPI) and examined under ultraviolet (UV) light for morphological nuclear changes of apoptosis. Representative fluorescent photomicrographs show a dose-dependent increase in condensed apoptotic nuclei (red arrows) with nearly all apoptotic nuclei at the highest dose (80 µM). (a) Representative phase-contrast micrographs show shrinkage and condensation of the cells. (b, c) HSC-T6 were plated and treated with the indicated doses of FC, an equivalent dose of NC or 0.1% DMSO (d) and untreated (U). Stellate cells were treated with 20, 40 and 80 µM of FC or NC. After treatment, cells were fixed with 3.7% paraformaldehyde, and stained with DAPI. In all, 200 DAPI-positive cells were counted for live and dead cells. The percentage of the dead cells to the total cells was calculated as the total number of cells divided by the number of dead cell multiplied by 100. (d) HSC-T6 were plated and treated with the indicated doses of FC, an equivalent dose of NanoCurc™ (NC), 0.1% DMSO (d) and untreated (U). Stellate cells were treated with 20, 40 and 80 µM of FC or NanoCurc™. After treatment, the cells were trypsinized without ethylenediaminetetraacetic acid (EDTA) combined with an equal volume of Trypan blue and counted on hemocytometer for live and dead cells per ml. Percentage of dead cells were calculated by dividing Trypan-positive cells by the total number of cells multiplied by 100. All experiments are representative from two experiments.