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Aim: The dual-ligand glycyrrhetinic acid and galactose-modified chitosan nanoparticles were designed to further improve the targeting capability to hepatocellular carcinoma (HCC). Materials & methods: The dual-ligand glycyrrhetinic acid and galactose-modified chitosan nanoparticles were fabricated by using ionic gelation method and their characteristics have been measured. Furthermore, the biodistribution and biocompatibility of this targeting vehicle were investigated in vitro and in vivo, respectively. Results: The targeting vehicle was specifically internalized into hepatoma cells in vitro and accumulated into tumor tissue in vivo with high efficacy. Moreover, the vehicle did not induce inflammation reaction and affect morphologies and organ functions. Conclusion: The targeting accumulation in HCC tissue and great biocompatibility of the dual-ligand modified chitosan nanoparticles highlight the potential of delivering anticancer agents into HCC cells.

As a gene therapy strategy, RNA interference (RNAi) offers tremendous tumor therapy potential. However, its therapeutic efficacy is restricted by its inferior ability for targeted delivery and cellular uptake of small interfering RNA (siRNA). This study sought to develop a dual-ligand nanoparticle (NP) system loaded with siRNA to promote targeted delivery and therapeutic efficacy. We synthesized a dual receptor-targeted chitosan nanosystem (GCGA), whose target function was controlled by the ligands of galactose of lactobionic acid (LA) and glycyrrhetinic acid (GA). By loading siPAK1, an siRNA targeting P21-activated kinase 1 (PAK1), a molecular-targeted therapeutic dual-ligand NP (GCGA–siPAK1) was established. We investigated the synergistic effect of these two targeting units in hepatocellular carcinoma (HCC). In particular, GCGA–siPAK1 enhanced the NP targeting ability and promoted siPAK1 cell uptake. Subsequently, dramatic decreases in cell proliferation, invasion, and migration, with an apparent increase in cell apoptosis, were observed in treated cells. Furthermore, this dual-ligand NP gene delivery system demonstrated significant anti-tumor effects in tumor-bearing mice. Finally, we illuminated the molecular mechanism, whereby GCGA–siPAK1 promotes endogenous cell apoptosis through the PAK1/MEK/ERK pathway. Thus, the dual-target property effectively promotes the HCC therapeutic effect and provides a promising gene therapy strategy for clinical applications.

ObjectiveThere is little evidence that adjuvant therapy after radical surgical resection of hepatocellular carcinoma (HCC) improves recurrence-free survival (RFS) or overall survival (OS). We conducted a multicentre, randomised, controlled, phase IV trial evaluating the benefit of an aqueous extract of Trametes robinophila Murr (Huaier granule) to address this unmet need.Design and resultsA total of 1044 patients were randomised in 2:1 ratio to receive either Huaier or no further treatment (controls) for a maximum of 96 weeks. The primary endpoint was RFS. Secondary endpoints included OS and tumour extrahepatic recurrence rate (ERR). The Huaier (n=686) and control groups (n=316) had a mean RFS of 75.5 weeks and 68.5 weeks, respectively (HR 0.67; 95% CI 0.55 to 0.81). The difference in the RFS rate between Huaier and control groups was 62.39% and 49.05% (95% CI 6.74 to 19.94; p=0.0001); this led to an OS rate in the Huaier and control groups of 95.19% and 91.46%, respectively (95% CI 0.26 to 7.21; p=0.0207). The tumour ERR between Huaier and control groups was 8.60% and 13.61% (95% CI −12.59 to −2.50; p=0.0018), respectively.ConclusionsThis is the first nationwide multicentre study, involving 39 centres and 1044 patients, to prove the effectiveness of Huaier granule as adjuvant therapy for HCC after curative liver resection. It demonstrated a significant prolongation of RFS and reduced extrahepatic recurrence in Huaier group.Trial registrationNCT01770431; Post-results.

Photosensitizer (PS) serves as the central element of photodynamic therapy (PDT). The use of common nanoparticles (NPs) for PDT has typically been rendered less effective by the undesirable aggregation-caused quenching (ACQ) effect, resulting in quenched fluorescence and reduced reactive oxygen species (ROS) generation that diminish the imaging quality and PDT efficacy. To overcome the ACQ effect and to enhance the overall efficacy of PDT, herein, integrin ανβ3-targeted organic nanodots for image-guided PDT were designed and synthesized based on a red emissive aggregation-induced emission (AIE) PS. Methods: The TPETS nanodots were prepared by nano-precipitation method and further conjugated with thiolated cRGD (cRGD-SH) through a click reaction to yield the targeted TPETS nanodots (T-TPETS nanodots). Nanodots were characterized for encapsulation efficiency, conjugation rate, particle size, absorption and emission spectra and ROS production. The targeted fluorescence imaging and antitumor efficacy of T-TPETS nanodot were evaluated both in vitro and in vivo. The mechanism of cell apoptosis induced by T-TPETS nanodot mediated-PDT was explored. The biocompatibility and toxicity of the nanodots was examined using cytotoxicity test, hemolysis assay, blood biochemistry test and histological staining. Results: The obtained nanodots show bright red fluorescence and highly effective 1O2 generation in aggregate state. Both in vitro and in vivo experiments demonstrate that the nanodots exhibit excellent tumor-targeted imaging performance, which facilitates image-guided PDT for tumor ablation in a hepatocellular carcinoma model. Detailed analysis reveals that the nanodot-mediated PDT is able to induce time- and concentration-dependent cell death. The use of PDT at a high PDT intensity leads to direct cell necrosis, while cell apoptosis via the mitochondria-mediated pathway is achieved under low PDT intensity. Conclusion: Our results suggest that well-designed AIE nanodots are promising for image-guided PDT applications.

Toll-like receptor (TLR) signaling has been implicated in inflammatory-related cancers. The upregulation of TLR signaling in hepatocellular carcinoma (HCC) suggests that it may play an essential role in the prognosis of chronic and inflammatory diseases that ultimately culminate in HCC. Here, we provide evidence about the involvement of the TLR pathway in the initiation, progression, and metastasis of HCC. The differential expression of TLR in epithelial cells has also been discussed. In particular, we emphasize the physiological role of TLR4 in the development and pathogenesis of HCC and propose novel and promising approaches for HCC therapeutics with the aid of TLR ligands.

Hepatocellular carcinoma (HCC) is the most common primary liver cancer with high morbidity and mortality worldwide. Chemotherapy is recommended to patients with intermediate or advanced stage cancer. However, the conventional chemotherapy yields low desired response rates due to multidrug resistance, fast clearance rate, nonspecific delivery, severe side effects, low drug concentration in cancer cells, and so on. Nanoparticle-mediated targeted drug delivery system can surmount the aforementioned obstacles through enhanced permeability and retention effect and active targeting as a novel approach of therapeutics for HCC in recent years. The active targeting is triggered by ligands on the delivery system, which recognize with and internalize into hepatoma cells with high specificity and efficiency. This review focuses on the latest targeted delivery systems for HCC and summarizes the ligands that can enhance the capacity of active targeting, to provide some insight into future research in nanomedicine for HCC.

Galactosylated chitosan (CTS) has been widely applied in liver tissue engineering as scaffold. However, the influence of degree of substitution (DS) of galactose moieties on cell attachment and mechanical stability is not clear. In this study, we synthesized the lactose-modified chitosan (Lact-CTS) with various DS of galactose moieties by Schiff base reaction and reducing action of NaBH4, characterized by FTIR. The DS of Lact-CTS-1, Lact-CTS-2, and Lact-CTS-3 was 19.66%, 48.62%, and 66.21% through the method of potentiometric titration. The cell attachment of hepatocytes on the CTS and Lact-CTS films was enhanced accompanied with the increase of galactose moieties on CTS chain because of the galactose ligand-receptor recognition; however, the mechanical stability of Lact-CTS-3 was reduced contributing to the extravagant hydrophilicity, which was proved using the sessile drop method. Then, the three-dimensional Lact-CTS scaffolds were fabricated by freezing-drying technique. The SEM images revealed the homogeneous pore bearing the favorable connectivity and the pore sizes of scaffolds with majority of 100 μm; however, the extract solution of Lact-CTS-3 scaffold significantly damaged red blood cells by hemolysis assay, indicating that exorbitant DS of Lact-CTS-3 decreased the mechanical stability and increased the toxicity. To sum up, the Lact-CTS-2 with 48.62% of galactose moieties could facilitate the cell attachment and possess great biocompatibility and mechanical stability, indicating that Lact-CTS-2 was a promising material for liver tissue engineering.

Restoration of blood flow to the ischemic liver lobes may paradoxically exacerbate tissue injury, which is called hepatic ischemia/reperfusion injury (IRI). Toll-like receptor 4 (TLR4), expressed on several liver cell types, and the nuclear factor-kappa B (NF-kappaB) signaling pathway are crucial to mediating hepatic inflammatory response. Because IRI is essentially a kind of profound acute inflammatory reaction evoked by many kinds of danger signals, we investigated TLR4/NF-kappaB signaling pathway activation in a murine model of partial hepatic IRI.Wild-type mice (WT, C3H/HeN) or TLR4 mutant mice (C3H/HeJ) were subjected to 45 minutes of partial hepatic ischemia followed by 1 hour, 3 hours of reperfusion. Sham group accepted the same procedure without the obstruction of blood supply. At the end of reperfusion, the compromise of liver function and the histological change of liver sections were measured as the severity of liver injury. The level of endotoxin in the portal vein was measured by limulus assay. NF-kappaB activation was determined by electrophoretic mobility shift assay (EMSA). The levels of tumor necrosis factor-alpha (TNF-alpha) and interleukin-1beta (IL-1beta) in systemic blood after hepatic IRI were assessed by enzyme-linked immunosorbent assay (ELISA).The compromise of liver function and the morphological injuries in mutant mice were relieved more markedly than those in WT mice after partial hepatic IRI. NF-kappaB activation in WT mice was stronger than that in TLR4 mutant mice, and both were stronger than those in the sham operated mice (P0.01). Endotoxin in each group was undetectable. The levels of TNF-alpha and IL-1beta in systemic blood were elevated in both strains, but lower in the sham operated group. These mediators were significantly decreased in TLR4 mutant mice compared with those in WT mice (P0.01).The TLR4/NF-kappaB signaling pathway may mediate hepatic IRI triggered by endogenous danger signals. Inhibition of the TLR4/NF-kappaB pathway may be a potential therapeutic target for attenuating ischemia/reperfusion-induced tissue damage in some clinical settings.

To investigate the curative effects of transplantation of hepatocytes differentiated from embryonic stem cells (ESCs) on treatment of fulminant hepatic failure (FHF).Mouse ESCs transfected with green fluorescent protein were cultured. RT-PCR was used to detect the mRNA expression of alpha-fetoprotein (AFP), transthyretin (TTR), hepatic nuclear factor (HNF), albumin (ALB), glucose-6-phosphate (G6P), and tyrosine aminotransferase (TAT) at different time points. Immunocytochemistry (ICC) was used to detect the expression of AFP, ALB, cytokeratin 8 (CK8), and CK11 in the cells. The morphology and distribution of the cells were observed with microscope. Forty mice were randomly divided into 2 equal groups: ESC transplantation group, undergoing transplantation of ICC positive ESCs (hepatocytes) into the hepatic capsule, and control group, undergoing injection of normal saline in the hepatic capsule. Twenty-four hours later carbon tetrachloride was injected intra-peritoneally to induce FHF. The survival status of the mice was observed. Twenty-four hours later venous blood samples were collected to examine the levels of total bilirubin (TB), alanine aminotransferase (ALT), ALB, blood sugar (BS), and prothrombin time (PT). After the death of the mice, their livers were taken out to undergo microscopy and immunohistochemistry to observe the structure of liver tissue, growth of tumor, and expression of ALB.RT-PCR showed that the mRNA expression of AFP, TTR, HNF, ALB, G6P, and TAT emerged since days 3, 3, 5, 9, and 11 respectively and then gradually increased till day 19. ICC showed that the EB cells began to express AFP since day 7, to express CK8 and CK11 since day 9, and to express ALB since day 11. The ICC-positive cells were consistent with the mouse hepatocytes morphologically and were distributed only in the central and marginal areas of the EB cell community. Injected with carbon tetrachloride, the mice showed manifestations of FHF. However, the symptoms of central nervous system emerged later in the mice implanted with the hepatocytes in comparison with the control mice; 8 of the 20 mice in the transplantation group showed ascites in comparison with 14 in the control group. The mean survival time of the transplantation group was 62 hours, significantly longer than that of the control group (23 hours, P0.05). Compare with the normal mice, the FHF mice in both groups showed higher ALT and TB and lower ALB and BS (all P0.01), however, the levels of ALT and TB were lower, the level of BS was higher, and PT was shorter significantly in the transplantation group than in the control group (all P0.05). Pathology showed that no tumor formation was found in both groups and that the transplanted hepatocytes were incorporated well into the liver parenchymal structure and expressed ALB.Hepatocytes originating from ESCs exercise the functions of normal hepatocytes. Transplantation of hepatocytes differentiated from ESCs is able to improve the life quality and lengthen the survival time of FHF mice.

To construct a eukaryotic expression vector carrying the small hairpin RNA (shRNA) for Toll-like receptor 4 (TLR4) mRNA and a reporter gene of enhanced green fluorescence protein (EGFP) and study the inhibition of cytokine release by rat RAW264.7 macrophages induced by lipopolysaccharide (LPS) stimulation through transfection and expression of shRNA targeting TLR4 gene via the RNAi mechanism.The H1 promotor and double BbsIrestrict endoenzyme site from the plasmid psiRNA-hH1neo were cloned into the reporter gene plasmid pEGFP-C1 at the MluIrestrict endoenzymic site, thus forming the plasmid pEGFP-H1/siRNA containing Bbs site and reporter EGFP gene. Then an oligo nuclear hairpin sequence targeting TLR4 gene was designed by the internet tool siRNA Wizard and then inserted into the plasmid pEGFP-H1/siRNA so as to form the plasmid pEGFP-H1/TLR4-siRNA. Rat macrophages of the line RAW264.7 were cultured and transfected with pEGFP-H1/TLR4-siRNA mediated by lipofectamine 2000. Another RAW264.7 cells were transfected with pEGFP-H1/control sequence-siRNA or blank plasmid. Lipopolysaccharide was added into the 3 kinds of culture fluid for 2 and 68 hours respectively. ELSA was used to detect the levels of tumor necrosis factor-alpha (TNF-alpha) in the supernatants.Restriction endonuclease analysis showed that the construction pEGFP-H1/TLR4-siRNA carrying hairpin RNA for TLR4 gene and reporter EGFP gene was successful. The expression of EGFP gene was 50% +/- 8%. The TNF-alpha level of the TLR4-siRNA transfection group 2 hours and after transfection was 825 pg/ml +/- 136 pg/ml, significantly lower than those of the pEGFP-H1/control sequence-siRNA and blank plasmid groups (2190 pg/ml +/- 359 pg/ml and 1265 pg/ml +/- 283 pg/ml respectively, both P0.01). The TNF-alpha level of the TLR4-siRNA transfection group 8 hours and after transfection was 1179 pg/ml +/- 240 pg/ml, significantly lower than those of the pEGFP-H1/control sequence-siRNA and blank plasmid groups (4720 pg/ml +/- 227 pg/ml and 4689 pg/ml +/- 310 pg/ml respectively, both P0.01).shRNA targeting TLR4 gene can inhibit the TNF-alpha release by RAW264.7 cells evoked by LPS.

To investigate the mechanism and regulation of differentiation from embryonic stem (ES) cells into biliary epithelium (BE) cells and to find a new source f of BE cells for liver engineering.ES cells of BALB/c mice (BALB/c-ES) and ES cells of mice of the line 129 (D3-ES) were cultured in the medium without LIF for 5 days with the result that embryoid bodies (EBs) were developed from the ES cells. For directional differentiation, the EBs were plated onto a 24-well gelatin-coated tissue culture dish and some growth factors, such as transforming growth factor (TGF), acid fibroblast growth factor (aFGF), hepatocyte growth factor (HGF) and epidermal growth factor (EGF) etc were added into the medium successively in the experiment group but not in the control group. The differentiation status was observed by inversion microscope dynamically. The BE cell markers, such as cytokeratin 7 (CK7), cytokeratin 19 (CK19) and gamma-glutamyltransferase (GGT) were detected by immunocytochemistry (ICC) and histochemistry.After the culture for 5 days the ES cells developed into many EBs in the medium without LIF. In the EBs cells clusters, many circular structures appeared on the differentiation day 10. On the days 10 and 13 ICC, CK7 and CK19 began to be expressed respectively. GGT began to be expressed at day 10. CK7, CK19 and GGT were also expressed in the ES cells of the control group, however, appeared remarkably later.ES cells can differentiate into BE cells under specific culture condition and the action of growth factors. Such ES cell differentiating system can provide BE cells and may serve as a good new source of differentiated cell types for liver engineering.

To evaluate the effect of laparoscopic spleen-preserving operation for traumatic spleen rupture.From 1997 to 2003, 15 cases of traumatic spleen rupture were treated with laparoscopic spleen-preserving operation in our hospital. Nine cases had operation history in the middle and lower abdomen. ZT binding, electrocoagulation, fibrin and gelfoam tamping and suture repairing were used in patients with spleen rupture of grade I and grade II. Combined hemostasis was used for spleen rupture of grade III.All patients did not need laparotomy during operation and no postoperative bleeding occurred. They were all cured and followed up for 3-12 months. Determination of immunoglobulins after operation showed normal, and spleen ultrasonic examination, CT and body state evaluations were all satisfactory.Laparoscopy in the management of spleen trauma can be used in confirmed diagnosis and in determining the degree of spleen injury. For patients with stable vital signs laparoscopic spleen-preserving operation can be used. The laparoscopic spleen-preserving operation is safe in the treatment of traumatic spleen rupture.

To investigate the mechanism and regulation of differentiation from embryonic stem (ES) cells to hepatocytes in vitro and to find a new source of cell types for hepatocytes replacement therapies in the treatment of hepatic failure.ES cells of BALB/c mice were cultured and maintained undifferentiated in gelatin-coated dishes in Dubecco's modified Eagle's medium (DMEM) containing 1 000 U/L leukaemia inhibitory factor (LIF). Then, the ES cells were cultured in DMEM without LIF so as to develop into embryonic bodies (EBs). For directional differentiating, EBs were put into 24-well tissue culture dish and differentiate into cells with different forms. On day 7 to day 11 acid fibroblast growth factor (aFGF) with terminal concentration of 100 ng/ml was added, and on day 11 to day 15 hepatocyte growth factor (HGF) terminal concentration of 20 ng/ml was added. EBs in culture without growth factor was used as controls. The development and differentiation status was observed by inversion microscope dynamically. The concentrations of alpha-fetoprotein (AFP) and albumin (ALB) in the supernatants were determined by radioimmunoassay on the 1st to 15th day since the ES cells were inoculated. Immunocytochemistry (ICC) was used to detect the expression of hepatic proteins such as AFP, ALB, cytokeratin 8 (CK8) and cytokeratin 18 (CK18) in the cells on the days 7, 8, 9, 10, 11, 12, 13, 14, and 15. The number of ALB positive cells was counted. The ALB positive rate was calculated as the differentiation ratio of hepatic cells.ES cells remained undifferentiated and grew to ES clones in the DMEM containing LIF and developed to EBs in the medium without LIF. AFP was detected firstly in the medium on day 8 with a concentration of 3.4 ng/ml and the concentration increased to be 22.8 ng/ml at day 15; ALB was detected firstly in the medium on day 11 with a concentration of 0.2 microg/ml and the concentration increased to be 2.2 microg/ml on day15. ICC showed that AFP, ALB, CK8, and CK18 began to be expressed on day 8, day 10, day 10, and day 11 respectively. The ICC-positive cells have the ultrastructures consistent with those of the normal mouse hepatocytes. The ratio of hepatic differentiation was 30%.ES cells can differentiate directionally into hepatocytes when cultured in gelatin-coated dish and medium with aFGF and HGF. This ES cell differentiating system provides enough functional hepatocytes and may be a good new source of differentiated cell types for hepatocytes replacement therapies in treatment of hepatic failure.

AIM: To investigate the safety, rationality and the practicality of enteral nutritional (EN) support in the postoperative patients with damaged liver function and the protective effect of EN on the gut barrier. METHODS: 135 patients with liver function of Child B or C grade were randomly allocated to enteral nutrition group (EN, 65 cases), total parenteral nutrition group (TPN, 40 cases) and control group (CON, 30 cases). Nutritional parameters, hepatic and kidney function indexes were measured at the day before operation, 5th and 10th day after the operation respectively. Comparison was made to evaluate the efficacy of different nutritional support. Urinary concentrations of lactulose (L) and mannitol (M) were measured by pulsed electrochemical detection (HPLC-PED) and the L/M ratio calculated to evaluate their effectiveness on protection of gut barrier. RESULTS: No significant damages in hepatic and kidney function were observed in both EN and TPN groups between pre- and postoperatively. EN group was the earliest one reaching the positive nitrogen balance after operation and with the lowest loss of body weight and there was no change in L/M ratio after the operation (0.026 ± 0.004) at the day 1 before operation, 0.030 ± 0.004 at the day 5 postoperative and 0.027 ± 0.005 at the day 10 postoperative), but the change in TPN group was significant at the day 5 postoperative (0.027 ± 0.003 vs 0.038 ± 0.009, P < 0.01). CONCLUSION: EN is a rational and effective method in patients with hepatic dysfunction after operation and has significant protection effect on the gut barrier.