Autologous bone marrow infusion via portal vein combined with splenectomy for decompensated liver cirrhosis: A retrospective study.

BACKGROUND: In a previous study, autologous bone marrow infusion (ABMI) was performed in patients with decompensated liver cirrhosis (DLC) and acquired immunodeficiency syndrome and achieved good results, but whether splenectomy affected outcome was unclear. AIM: To investigate the efficacy of ABMI combined with splenectomy for treatment of DLC. METHODS: Eighty-three patients with DLC were divided into an intervention group (43 cases) and control group (40 cases) according to whether splenectomy was performed. The control group was treated with ABMI through the right omental vein, and the intervention group was additionally treated with splenectomy. RESULTS: After ABMI, the prothrombin time, serum total bilirubin levels, ascites volume and model for end-stage liver disease score in both groups were significantly lower, while the albumin levels were significantly higher than before ABMI (P < 0.01), but there were no significant differences between the groups (P > 0.05). After ABMI, the white blood cell and platelets counts in both groups were significantly higher than before ABMI (P < 0.01), and the counts in the intervention group were significantly higher than in the control group (P < 0.01). After ABMI the CD4+ and CD8+ T cell counts in both groups were significantly higher than before ABMI (P < 0.01). The CD8+ T cell counts in the intervention group increased continuously and the increase had a shorter duration compared with control group. CONCLUSION: ABMI through the portal vein in patients with DLC can significantly improve liver synthetic and secretory functions, and splenectomy promotes improvement of bone marrow hematopoietic and cellular immune functions.

Therapeutic effect of autologous bone marrow cells injected into the liver under the guidance of B­ultrasound in the treatment of HBV­related decompensated liver cirrhosis.

The 5-year mortality rates associated with decompensated liver cirrhosis (DLC) can reach 50%, which suggests that this condition poses a serious health risk. In previous studies conducted by our group, autologous bone marrow nucleated cells (ABMNCs) were used to treat HIV-positive patients with DLC through the right omental vein; however, trauma and poor compliance were encountered. In the present study, the percutaneous liver approach to inject ABMNCs under the guidance of B-ultrasound was employed for the treatment of DLC. A total of 108 patients with DLC were retrospectively divided into the routine drug treatment group (control group; 30 cases), the right omental vein infusion of ABMNCs group (observation group 1; 38 cases) and the B-ultrasound-guided liver injection of ABMNCs group (observation group 2; 40 cases). After treatment, the liver synthesis (prothrombin time, albumin and ascites) and secretion functions (total bilirubin) in observation groups 1 and 2 were significantly improved compared with those of the control group (P<0.01) and the bone marrow function was also significantly improved compared with that of the control group (P<0.01). While, the bone marrow function (white blood cell, platelet, and hemoglobin) in observation group 1 was significantly improved compared with that of observation group 2 at the end of treatment (P<0.01). After a 1-year follow-up, the case fatality rate was 2.5% (1/40) in observation group 2, which was significantly lower than the 20% fatality rate (6/30) recorded in the control group (P<0.05). The injection of ABMNCs into the liver under the guidance of B-ultrasound was significantly better than conventional drug therapy in treating DLC. This approach has obvious advantages such as no hospitalization, minimal trauma, rapid recovery and good compliance, all of which make it worthy of application in primary hospitals.

Inhibitory of active dual cancer targeting 5-Fluorouracil nanoparticles on liver cancer in vitro and in vivo.

The chitosan (CS) material as the skeleton nano-drug delivery system has the advantages of sustained release, biodegradability, and modifiability, and has broad application prospects. In the previous experiments, biotin (Bio) was grafted onto CS to synthesize biotin-modified chitosan (Bio-CS), and it was confirmed that it has liver cancer targeting properties. Single-targeted nanomaterials are susceptible to pathological and physiological factors, resulting in a state of ineffective binding between ligands and receptors, so there is still room for improvement in the targeting of liver cancer. Based on the high expression of folate (FA) receptors on the surface of liver cancers, FA was grafted onto Bio-CS by chemical synthesis to optimize the synthesis of folic acid-modified biotinylated chitosan (FA-CS-Bio), verified by infrared spectroscopy and hydrogen-1 nuclear magnetic resonance spectroscopy. The release of FA-CS-Bio/fluorouracil (5-FU) had three obvious stages: fast release stage, steady release stage, and slow release stage, with an obvious sustained release effect. Compared with Bio-CS, FA-CS-Bio could promote the inhibition of the proliferation and migration of liver cancer by 5-FU, and the concentration of 5-FU in hepatoma cells was significantly increased dose-dependently. Laser confocal experiments confirmed that FA-CS-Bio caused a significant increase in the fluorescence intensity in liver cancer cells. In terms of animal experiments, FA-CS-Bio increased the concentration of 5-FU in liver cancer tissue by 1.6 times on the basis of Bio-CS and the number of monophotons in liver cancer tissue by in vivo dynamic imaging experiments was significantly stronger than that of Bio-CS, indicating that the targeting ability of FA-CS-Bio was further improved. Compared with Bio-CS, FA-CS-Bio can significantly prolong the survival time of 5-FU in the orthotopic liver cancer transplantation model in mice, and has a relieving effect on liver function damage and bone marrow suppression caused by 5-FU. In conclusion, FA-CS-Bio nanomaterials have been optimized for synthesis. In vivo and in vitro experiments confirmed that FA-CS-Bio can significantly improve the targeting of liver cancer compared with Bio-CS. FA-CS-Bio/5-FU nanoparticles can improve the targeted inhibition of the proliferation and migration of liver cancer cells, prolong the survival period of tumor-bearing mice, and alleviate the toxic and side effects.

Autologous Bone Marrow Cell Infusion for the Treatment of Decompensated Liver Cirrhosis Patients With Type 2 Diabetes Mellitus.

This study aimed to indicate whether autologous bone marrow cell infusion (ABMI) via the right omental vein (ROV) could have a regulatory effect on decompensated liver cirrhosis (DLC) patients with type 2 diabetes mellitus (T2DM). For this purpose, 24 DLC patients with T2DM were divided into observation group (n=14) and control group (n=10). Patients in the observation group were given ABMI through the ROV and right omental artery (ROA), and cases in the control group received ABMI through the ROV. At 1, 3, 6, and 12months after ABMI, it was revealed that the prothrombin time, the total bilirubin levels, and the amount of ascites were significantly lower, while the serum albumin levels in the two groups were markedly higher compared with those before ABMI (p<0.01), and there was no significant difference between the two groups at each time point (p>0.05). The fasting blood glucose and glycosylated hemoglobin levels at 6 and 12months after ABMI in the two groups significantly decreased compared with those before ABMI (p<0.05 or p<0.01), while the decreased levels in the observation group were more obvious than those in the control group at each time point (p<0.01). The amount of insulin in the observation group at 3, 6, and 12months after ABMI was significantly less than that before ABMI in the control group (p<0.01). In summary, ABMI showed a significant therapeutic efficacy for DLC patients with T2DM through ROV and ROA.

Preoperative antiviral therapy and microvascular invasion in hepatitis B virus-related hepatocellular carcinoma: A meta-analysis.

Microvascular invasion (MVI) is an important predictor of metastatic tumour recurrence and is associated with adverse outcomes and poor prognosis in Hepatitis B virus (HBV)-related hepatocellular carcinoma (HCC) patients. The association between varying regimens of anti-viral drugs with the incidence of MVI in HBV-related HCC has been demonstrated, however, no meta-analysis of the available data has been conducted. Therefore, the current study sought to evaluate the association of preoperative antiviral therapy with incidence of microvascular invasion in HCC hepatitis virus patients. A systematic search of the literature was performed in MEDLINE/PubMed, Web of Science (WoS), and Scopus, up to January 2020. A random-effects model was used to estimate pooled odds ratios (ORs). Overall, six studies, with 4988 patients, met our inclusion criteria. The pooled OR of MVI in the patients who had preoperative antiviral therapy versus the patients who did not have antiviral therapy was; OR: 0.60, 95% Confidence Interval (CI): 0.49-0.73; I2 = 25%. In this study, a significant reduction in the OR of MVI was evident in patients who had anti-viral therapy.

Autologous bone marrow cell transplantation in the treatment of HIV patients with compensated cirrhosis.

Liver stem cell therapy is a promising tool to improve decompensated liver cirrhosis (DLC). Especially in patients infected with human immunodeficiency virus (HIV), the condition of the liver may be aggravated by antiretroviral therapy. A total of 21 patients diagnosed with DLC and HIV infection were divided into two groups as follows: those who received (combination therapy group, 14 patients) and those who did not receive (routine therapy group, 7 patients) bone marrow cell transplantation through the portal vein. Two patients died of surgery-related complications in the combination therapy group. The results showed that the survival rate was 85.7% in the combination therapy group after 2 years of follow-up, which was significantly higher than the 14.3% in the conventional therapy group (P<0.01). After treatment, the liver function score decreased significantly in the combination therapy group at 1 (t = 4.276, P = 0.000), 3 (t = 9.153, P = 0.000), and 12 (t = 13.536, P = 0.000) months, the levels of albumin were significantly increased, and the total bilirubin level and prothrombin time were significantly reduced or shortened as compared with the routine therapy group (P<0.05 or <0.01). The white blood cell count, hemoglobin, platelet count, and CD4+ and CD8+ levels were significantly higher in the combination therapy group at different time points as compared with the routine therapy group (P<0.05 or <0.01). In summary, the combination therapy is effective in HIV-infected patients with DLC and useful for the recovery of liver function and cellular immune function but may increase the risk of severe complications after surgery.

Synthesis of Biotin-Modified Galactosylated Chitosan Nanoparticles and Their Characteristics in Vitro and in Vivo.

BACKGROUND/AIMS: Our previous study found that a nanoparticle drug delivery system that operates as a drug carrier and controlled release system not only improves the efficacy of the drugs but also reduces their side effects. However, this system could not efficiently target hepatoma cells. The aim of this study was to synthesize biotin-modified galactosylated chitosan nanoparticles (Bio-GC) and evaluate their characteristics in vitro and in vivo. METHODS: Bio-GC nanomaterials were synthesized, and confirmed by fourier transform infrared spectroscopy (FT-IR) and hydrogen-1 nuclear magnetic resonance (1H-NMR). The liver position and cancer target property of Bio-GC nanoparticles in vitro and in vivo was tested by confocal laser and small animal imaging system. The characteristics of Bio-GC/5-fluorouracil (5-FU) nanoparticles in vitro and in vivo were explored by cell proliferation, migration and cytotoxicity test, or by animal experiment. RESULTS: Bio-GC nanoparticles were synthesized with biodegradable chitosan as the nanomaterial skeleton with biotin and galactose grafts. Bio-GC was confirmed by FT-IR and 1H-NMR. Bio-GC/5-FU nanoparticles were synthesized according to the optimal mass ratio for Bio-GC/5-FU (1: 4) and had a mean particle size of 81.1 nm, zeta potential of +39.2 mV, and drug loading capacity of 8.98%. Bio-GC/5-FU nanoparticles had sustained release properties (rapid, steady, and slow release phases). Bio-GC nanoparticles targeted liver and liver cancer cell in vitro and in vivo, and this was confirmed by confocal laser scanning and small animal imaging system. Compared with GC/5-FU nanoparticles, Bio-GC/5-FU nanoparticles showed more specific cytotoxic activity in a dose- and time-dependent manner and a more obvious inhibitory effect on the migration of liver cancer cells. In addition, Bio-GC/5-FU nanoparticles significantly prolonged the survival time of mice in orthotopic liver cancer transplantation model compared with other 5-FU nanoparticles or 5-FU alone. Bio-GC (0.64%) nanomaterial had no obvious cytotoxic effects on cells; thus, the concentration of Bio-GC/5-FU nanoparticles used was only 0.04% and showed no toxic effects on the cells. CONCLUSION: Bio-GC is a liver- and cancer-targeting nanomaterial. Bio-GC/5-FU nanoparticles as drug carriers have stronger inhibitory effects on the proliferation and migration of liver cancer cells compared with 5-FU in vitro and in vivo.

Anti-cancer efficacy of biotinylated chitosan nanoparticles in liver cancer.

The present study investigated the synthesis of biotinylated chitosan (Bio-CS) from chitosan using a nanomaterial skeleton with biotin and the successful targeting of the formulation in liver cancer cells. Bio-CS was validated by fourier transformed infrared spectroscopy and hydrogen-1 nuclear magnetic resonance spectroscopy. Bio-CS and plasmid DNA were used to construct Bio-CS/plasmid DNA nanoparticles according to the optimal molar ratio of 1:1 and the optimal pH-value of 5.5. Under these conditions, the parameters mean particle size, potential, encapsulation rate and drug loading, were 82.9 nm, +21.8 mV, 85.7% and 35.4%, respectively. Bio-CS exhibited an apparent liver cancer targeting effect in vitro and in vivo, as demonstrated by confocal laser scanning, green fluorescent protein transfection, and in vivo imaging assays. In addition, the Bio-CS/plasmid DNA nanoparticles significantly increased the survival period of the orthotropic liver cancer mouse model compared with the plasmid DNA, with no apparent side effects on the cells. Bio-CS nanomaterials stimulated an immune response in hepatoma cells via increased expression of GM-CSF, IL-21 and Rae-1 markers. The data suggest that Bio-CS increased the inhibition of liver cancer cell proliferation in vitro and the activation of the cellular immunity in vivo.

Proteasome inhibitor MG132 potentiates TRAIL-induced apoptosis in gallbladder carcinoma GBC-SD cells via DR5-dependent pathway.

TRAIL is a tumor-selective apoptosis-inducing cytokine playing a vital role in the surveillance and elimination of some tumor cells. However, some tumors are resistant to TRAIL treatment. Proteasome inhibitor MG132 exhibits anti-proliferative and pro-apoptotic properties in many tumors. In this study, we demonstrated that proteasome inhibitor MG132 in vitro and in vivo potentiates TRAIL-induced apoptosis in gallbladder carcinoma GBC-SD cells. MG132 was able to inhibit the proliferation of GBC-SD cells and induce apoptosis in a dose-dependent manner. The induction of apoptosis by proteasome inhibitor MG132 was mainly through the extrinsic apoptotic pathways of caspase activation such as caspase-8, caspase-3 and PARP cleavage. In addition, this process was also dependent on the upregulation of death receptor 5 (DR5), which promoted TRAIL-induced apoptosis in GBC-SD cells. Taken together, these findings indicate that MG132 possesses anti-gallbladder cancer potential that correlate with regulation of DR5-dependent pathway, and suggest that MG132 may be a promising agent for sensitizing GBC-SD cells to TRAIL-induced apoptosis.

Optimized synthesis of glycyrrhetinic acid-modified chitosan 5-fluorouracil nanoparticles and their characteristics.

The nanoparticle drug delivery system, which uses natural or synthetic polymeric material as a carrier to deliver drugs to targeted tissues, has a broad prospect for clinical application for its targeting, slow-release, and biodegradable properties. Here, we used chitosan (CTS) and hepatoma cell-specific binding molecule glycyrrhetinic acid to synthesize glycyrrhetinic acid-modified chitosan (GA-CTS). The synthetic product was confirmed by infrared (IR) spectra and hydrogen-1 nuclear magnetic resonance. The GA-CTS/5-fluorouracil (5-FU) nanoparticles were synthesized by combining GA-CTS and 5-FU and conjugating 5-FU onto the GA-CTS nanomaterial. The central composite design was performed to optimize the preparation process as CTS:tripolyphosphate sodium (TPP) weight ratio =5:1, 5-FU:CTS weight ratio =1:1, TPP concentration =0.05% (w/v), and cross-link time =50 minutes. GA-CTS/5-FU nanoparticles had a mean particle size of 193.7 nm, a polydispersity index of 0.003, a zeta potential of +27.4 mV, and a drug loading of 1.56%. The GA-CTS/5-FU nanoparticle had a protective effect on the drug against plasma degrading enzyme, and provided a sustained release system comprising three distinct phases of quick, steady, and slow release. Our study showed that the peak time, half-life time, mean residence time and area under the curve of GA-CTS/5-FU were longer or more than those of the 5-FU group, but the maximum concentration (C(max)) was lower. We demonstrated that the nanoparticles accumulated in the liver and have significantly inhibited tumor growth in an orthotropic liver cancer mouse model.

Activation of cellular immunity and marked inhibition of liver cancer in a mouse model following gene therapy and tumor expression of GM-SCF, IL-21, and Rae-1.

BACKGROUND: Cancer is both a systemic and a genetic disease. The pathogenesis of cancer might be related to dampened immunity. Host immunity recognizes nascent malignant cells - a process referred to as immune surveillance. Augmenting immune surveillance and suppressing immune escape are crucial in tumor immunotherapy. METHODS: A recombinant plasmid capable of co-expressing granulocyte-macrophage colony- stimulating factor (GM-SCF), interleukin-21 (IL-21), and retinoic acid early transcription factor-1 (Rae-1) was constructed, and its effects determined in a mouse model of subcutaneous liver cancer. Serum specimens were assayed for IL-2 and INF-γ by ELISA. Liver cancer specimens were isolated for Rae-1 expression by RT-PCR and Western blot, and splenocytes were analyzed by flow cytometry. RESULTS: The recombinant plasmid inhibited the growth of liver cancer and prolonged survival of tumor-loaded mice. Activation of host immunity might have contributed to this effect by promoting increased numbers and cytotoxicity of natural killer (NK) cells and cytotoxic T lymphocytes (CTL) following expression of GM-SCF, IL-21, and Rae-1. By contrast, the frequency of regulatory T cells was decreased, Consequently, activated CTL and NK cells enhanced their secretion of INF-γ, which promoted cytotoxicity of NK cells and CTL. Moreover, active CTL showed dramatic secretion of IL-2, which stimulates CTL. The recombinant expression plasmid also augmented Rae-1 expression by liver cancer cells. Rae-1 receptor expressing CTL and NK cells removed liver cancer. CONCLUSIONS: The recombinant expression plasmid inhibited liver cancer by a mechanism that involved activation of cell-mediated immunity and Rae-1 in liver cancer.

Synthesis of liver-targeting dual-ligand modified GCGA/5-FU nanoparticles and their characteristics in vitro and in vivo.

Nanoparticle drug delivery systems using polymers hold promise for clinical applications. We synthesized dual-ligand modified chitosan (GCGA) nanoparticles using lactic acid, glycyrrhetinic acid, and chitosan to target the liver in our previous studies. We then synthesized the GCGA/5-FU nanoparticles by conjugating 5-fluorouracil (5-FU) onto the GCGA nanomaterial, which had a mean particle size of 239.9 nm, a polydispersity index of 0.040, a zeta potential of +21.2 mV, and a drug loading of 3.90%. GCGA/5-FU nanoparticles had good slow release properties, and the release process could be divided into five phases: small burst release, gentle release, second burst release, steady release, and slow release. Inhibitory effects of GCGA/5-FU on tumor cells targeted the liver, and were time and dose dependent. GCGA nanoparticles significantly prolonged the efficacy of 5-FU on tumor cells, and alleviated the resistance of tumor cells to 5-FU. GCGA/5-FU nanoparticles were mostly concentrated in the liver, indicating that the GCGA nanoparticles were liver targeting. GCGA/5-FU nanoparticles significantly suppressed tumor growth in orthotopic liver transplantation mouse model, and improved mouse survival.

Glycyrrhetinic acid-modified chitosan nanoparticles enhanced the effect of 5-fluorouracil in murine liver cancer model via regulatory T-cells.

Modified chitosan nanoparticles are a promising platform for drug, such as 5-fluorouracil (5-FU), gene, and vaccine delivery. Here, we used chitosan and hepatoma cell-specific binding molecule glycyrrhetinic acid (GA) to synthesize glycyrrhetinic acid-modified chitosan (GA-CTS). The synthetic product was confirmed by infrared spectroscopy and hydrogen nuclear magnetic resonance. By combining GA-CTS and 5-FU, we obtained a GA-CTS/5-FU nanoparticle, with a particle size of 193.7 nm, drug loading of 1.56%, and a polydispersity index of 0.003. The GA-CTS/5-FU nanoparticle provided a sustained-release system comprising three distinct phases of quick, steady, and slow release. In vitro data indicated that it had a dose- and time-dependent anticancer effect. The effective drug exposure time against hepatic cancer cells was increased in comparison with that observed with 5-FU. In vivo studies on an orthotropic liver cancer mouse model demonstrated that GA-CTS/5-FU significantly inhibited cancer cell proliferation, resulting in increased survival time. The antitumor mechanisms for GA-CTS/5-FU nanoparticle were possibly associated with an increased expression of regulatory T-cells, decreased expression of cytotoxic T-cell and natural killer cells, and reduced levels of interleukin-2 and interferon gamma.

Synthesis of glycyrrhetinic acid-modified chitosan 5-fluorouracil nanoparticles and its inhibition of liver cancer characteristics in vitro and in vivo.

Nanoparticle drug delivery (NDDS) is a novel system in which the drugs are delivered to the site of action by small particles in the nanometer range. Natural or synthetic polymers are used as vectors in NDDS, as they provide targeted, sustained release and biodegradability. Here, we used the chitosan and hepatoma cell-specific binding molecule, glycyrrhetinic acid (GA), to synthesize glycyrrhetinic acid-modified chitosan (GA-CTS). The synthetic product was confirmed by Fourier transformed infrared spectroscopy (FT-IR) and ¹H-nuclear magnetic resonance (¹H-NMR). By combining GA-CTS and 5-FU (5-fluorouracil), we obtained a GA-CTS/5-FU nanoparticle, with a particle size of 217.2 nm, a drug loading of 1.56% and a polydispersity index of 0.003. The GA-CTS/5-FU nanoparticle provided a sustained release system comprising three distinct phases of quick, steady and slow release. We demonstrated that the nanoparticle accumulated in the liver. In vitro data indicated that it had a dose- and time-dependent anti-cancer effect. The effective drug exposure time against hepatic cancer cells was increased in comparison with that observed with 5-FU. Additionally, GA-CTS/5-FU significantly inhibited the growth of drug-resistant hepatoma, which may compensate for the drug-resistance of 5-FU. In vivo studies on an orthotropic liver cancer mouse model demonstrated that GA-CTS/5-FU significantly inhibited tumor growth, resulting in increased survival time.

Galactosylated chitosan/5-fluorouracil nanoparticles inhibit mouse hepatic cancer growth and its side effects.

AIM: To observe the curative effect of galactosylated chitosan (GC)/5-fluorouracil (5-FU) nanoparticles in liver caner mice and its side effects. METHODS: The GC/5-FU nanoparticle is a nanomaterial made by coupling GC and 5-FU. The release experiment was performed in vitro. The orthotropic liver cancer mouse models were established and divided into control, GC, 5-FU and GC/5-FU groups. Mice in the control and GC group received an intravenous injection of 200 µL saline and GC, respectively. Mice in the 5-FU and GC/5-FU groups received 200 µL (containing 0.371 mg 5-FU) 5-FU and GC/5-FU, respectively. The tumor weight and survival time were observed. The cell cycle and apoptosis in tumor tissues were monitored by flow cytometry. The expression of p53, Bax, Bcl-2, caspase-3 and poly adenosine 50-diphosphate-ribose polymerase 1 (PARP-1) was detected by immunohistochemistry, reverse transcription-polymerase chain reaction and Western blot. The serum blood biochemical parameters and cytotoxic activity of natural killer (NK) cell and cytotoxicity T lymphocyte (CTL) were measured. RESULTS: The GC/5-FU nanoparticle is a sustained release system. The drug loading was 6.12% ± 1.36%, the encapsulation efficiency was 81.82% ± 5.32%, and the Zeta potential was 10.34 ± 1.43 mV. The tumor weight in the GC/5-FU group (0.4361 ± 0.1153 g vs 1.5801 ± 0.2821 g, P < 0.001) and the 5-FU (0.7932 ± 0.1283 g vs 1.5801 ± 0.2821 g, P < 0.001) was significantly lower than that in the control group; GC/5-FU treatment can significantly lower the tumor weight (0.4361 ± 0.1153 g vs 0.7932 ± 0.1283 g, P < 0.001), and the longest median survival time was seen in the GC/5-FU group, compared with the control (12 d vs 30 d, P < 0.001), GC (13 d vs 30 d, P < 0.001) and 5-FU groups (17 d vs 30 d, P < 0.001). Flow cytometry revealed that compared with the control, GC/5-FU caused a higher rate of G0-G1 arrest (52.79% ± 13.42% vs 23.92% ± 9.09%, P = 0.014 ) and apoptosis (2.55% ± 1.10% vs 11.13% ± 11.73%, P < 0.001) in hepatic cancer cells. Analysis of the apoptosis pathways showed that GC/5-FU upregulated the expression of p53 at both the protein and the mRNA levels, which in turn lowered the ratio of Bcl-2/Bax expression; this led to the release of cytochrome C into the cytosol from the mitochondria and the subsequent activation of caspase-3. Upregulation of caspase-3 expression decreased the PARP-1 at both the mRNA and the protein levels, which contributed to apoptosis. 5-FU increased the levels of aspartate aminotransferase and alanine aminotransferase, and decreased the numbers of platelet, white blood cell and lymphocyte and cytotoxic activities of CTL and NK cells, however, there were no such side effects in the GC/5-FU group. CONCLUSION: GC/5-FU nanoparticles can significantly inhibit the growth of liver cancer in mice via the p53 apoptosis pathway, and relieve the side effects and immunosuppression of 5-FU.

5-Fluorouracil nanoparticles inhibit hepatocellular carcinoma via activation of the p53 pathway in the orthotopic transplant mouse model.

Biodegradable polymer nanoparticle drug delivery systems provide targeted drug delivery, improved pharmacokinetic and biodistribution, enhanced drug stability and fewer side effects. These drug delivery systems are widely used for delivering cytotoxic agents. In the present study, we synthesized GC/5-FU nanoparticles by combining galactosylated chitosan (GC) material with 5-FU, and tested its effect on liver cancer in vitro and in vivo. The in vitro anti-cancer effects of this sustained release system were both dose- and time-dependent, and demonstrated higher cytotoxicity against hepatic cancer cells than against other cell types. The distribution of GC/5-FU in vivo revealed the greatest accumulation in hepatic cancer tissues. GC/5-FU significantly inhibited tumor growth in an orthotropic liver cancer mouse model, resulting in a significant reduction in tumor weight and increased survival time in comparison to 5-FU alone. Flow cytometry and TUNEL assays in hepatic cancer cells showed that GC/5-FU was associated with higher rates of G0-G1 arrest and apoptosis than 5-FU. Analysis of apoptosis pathways indicated that GC/5-FU upregulates p53 expression at both protein and mRNA levels. This in turn lowers Bcl-2/Bax expression resulting in mitochondrial release of cytochrome C into the cytosol with subsequent caspase-3 activation. Upregulation of caspase-3 expression decreased poly ADP-ribose polymerase 1 (PARP-1) at mRNA and protein levels, further promoting apoptosis. These findings indicate that sustained release of GC/5-FU nanoparticles are more effective at targeting hepatic cancer cells than 5-FU monotherapy in the mouse orthotropic liver cancer mouse model.

Preliminary pharmacology of galactosylated chitosan/5-fluorouracil nanoparticles and its inhibition of hepatocellular carcinoma in mice.

Biodegradable polymer nanoparticle drug delivery systems are characterized by targeted drug delivery, improved pharmacokinetic and biodistribution, enhanced drug stability and lowered side effects; these drug delivery systems are widely used for delivery of cytotoxic agents. The galactosylated chitosan (GC)/5-fluorouracil (5-FU) nanoparticle is a nanomaterial made by coupling GC, a polymer known to have the advantages described above, and 5-FU. The GC/5-FU nanoparticle is a sustained release system, it was showed that the peak time, half-life time, mean residence time (MRT) and area of under curve (AUC) of GC/5-FU were longer or more than those of the 5-FU group, but the maximum concentration (Cmax) was lower. The distribution of GC/5-FU in vivo revealed the greatest accumulation in the hepatic cancer tissues, and the hepatic cell was the target of the nanoparticles. Toxicology research showed that the toxicity of GC-5-FU was lower than that of 5-FU in mice. In vivo experiments showed that GC/5-FU can significantly inhibit tumor growth in an orthotropic liver cancer mouse model. GC/5-FU treatment can significantly lower the tumor weight and increase the survival time of mice when compared with 5-FU treatment alone. Flow cytometry and the TUNEL assay revealed that compared with 5-FU, GC/5-FU caused higher rates of G 0-G 1 arrest and apoptosis in hepatic cancer cells.

Synthesis of galactosylated chitosan/5-fluorouracil nanoparticles and its characteristics, in vitro and in vivo release studies.

Biodegradable polymer nanoparticle drug delivery systems are characterized by targeted drug delivery, improved pharmacokinetic and biodistribution, enhanced drug stability, and lowered side effects; these drug delivery systems are widely used for delivery of cytotoxic agents. The galactosylated chitosan (GC)/5-fluorouracil (5-FU) nanoparticle is a nanomaterial made by coupling GC, a polymer known to have the advantages described above, and 5-FU. We found that when 5-FU and GC were mixed at the mass ratio of 10:1, the nanoparticle reached a maximum encapsulation efficiency of 81.82% ± 5.32%, with a drug loading of 6.12% ± 1.36%, a particle size of 35.19 ± 9.50 nm, and a Zeta potential of +10.34 ± 1.43 mV. The GC/5-FU nanoparticle is a sustained release system, whose anticancer effects were shown to be dose and time dependent, with a higher cytotoxicity to hepatic cancer than to other cell types. The distribution of GC/5-FU in vivo revealed the greatest accumulation in the hepatic cancer tissues, with an 8.69-, 23.35-, 79.96-, and 85.15-fold increase when compared to normal liver tissue, kidney, heart and blood, respectively, suggesting that the hepatic cell was the target of the nanoparticles. In vivo experiments showed that GC/5-FU can significantly inhibit tumor growth in an orthotropic liver cancer mouse model. GC/5-FU treatment can significantly lower the tumor weight and increase the survival time of mice when compared to 5-FU treatment alone. Flow cytometry revealed that compared to 5-FU, GC/5-FU caused higher rates of G0-G1 arrest and apoptosis in hepatic cancer cells.

Design and synthesis of dual-ligand modified chitosan as a liver targeting vector.

Vector plays an important role in hepatic targeted drug delivery system. In this study, a novel material as a liver targeting vector, dual-ligand modified chitosan (GCGA) composed of chitosan (CTS), glycyrrhetinic acid (GA) and lactobionic acid (LA), was designed and synthesized by an orthogonal experiment with two-step synthesis under mild conditions. The synthesized final product was characterized and confirmed by FTIR and (1)H-NMR spectroscopy, and DS of GA and LA in CTS were measured to be 13.77 and 16.74 mol% using (1)H-NMR, respectively. The cytotoxicity of CTS and GCGA was concentration dependent which was inverse proportion to the cell viability by MTT assay using L929 cell line, and inhibitory concentration 50% (IC50) was 0.2 mg/ml for GCGA. The in vitro targeting efficiency and the in vitro cellular uptake were investigated. Compared with CTS NPs and GA-CTS NPs, GCGA NPs showed good cell specificity to BEL-7402 cells via the dual-ligand-receptor-mediated recognition, leading to a higher affinity to BEL-7402 cells. The results suggested that GCGA described here has the potential to be used as an effective vector for hepatic targeted drug therapy.

Synthesis and efficient hepatocyte targeting of galactosylated chitosan as a gene carrier in vitro and in vivo.

While chitosan (CS) has been researched widely as a non-viral vector, its usefulness has been limited by its low cell specificity and transfection efficiency. Therefore, we successfully synthesized galactosylated chitosan (GC) and complexed it with an enhanced green fluorescent protein plasmid (pIRES-EGFP) for transfection into cultured H22 cells (murine hepatic cancer cell line) using various GC/EGFP (N/P) charge ratios. Maximal gene transfection rates detected by flow cytometry occurred at an N/P ratio 5:1. Compared with those of lipofectin/EGFP and naked pIRES-EGFP, GC/EGFP complexes show a very efficient cell-selective transfection to hepatocytes. The MTT assay detected relatively low cytotoxicity in cells transfected with GC. A recombinant plasmid granulocyte-macrophage colony-stimulating factor (GM-SCF) and interleukin (IL) 21 (pIRES/GM-CSF-IL21) was successfully constructed and GC/GM-CSF-IL21 nanoparticles (average diameter, 82.1 nm) were administered via the tail vein of mice with liver metastasis of colon cancer model, for 5 consecutive days. The GC/GM-CSF-IL21 nanoparticles exhibited hepatocyte and passive tumor specificity, increased therapeutic efficacy compared to control groups, promoted leukocytes to aggregate in tumor tissues, and activated the cytotoxicity of natural killer (NK) cells and cytolytic T lymphocyte (CTL). Our results indicate that GC can be used in gene therapy to improve transfection efficiency and can be used as an immunological stimulant in vivo.