Acute hepatic failure (AHF) is a fulminant and complicated hepatic damage with organ failure, elevated hepatic enzymes, and finally causes hepatic encephalopathy. It is a big challenge to treat severe AHF in medical treament.1,2 Although liver transplantation provides a curative treatment for hepatic failure, there are still many limitations for hepatic transplant treatment.2–6 Stem cell therapeutics are the promising way for treating hepatic diseases.7–9 Pluripotent stem cells including embryonic stem cells and induced pluripotent stem cells can produce mature liver cells.10–13 However, limitation of good biosources and biocellular products for delivering the targets poses the major difficulty for treating the damaged liver and further facilitating hepatic regeneration.
Nonviral gene delivery, such as the use of biodegradable nanoparticles, has been considered a potentially safer gene-delivery method in comparison to conventional virus-based systems.14–17 The multi-step processing including polycationization of DNA-complexities could protect DNA degradation, and these DNA complexes are small enough to facilitate the cells maturing into intracellular organs.15–19 The novel particles, polymers, and polycomplex can significantly and efficiently bind to the cell membrane and intracellular organs.16–19 Cationic nanoparticles have been shown to be noncytotoxic and are characterized by high transfection efficiency, which makes them a promising in vivo gene-delivery material for disease treatment. It has been reported that cationic nanoparticle-mediated delivery of miR-145 and Oct4 with Sirt1 in mice suppressed lung adenocarcinoma tumor progression and promoted functional recovery of aged retinas, respectively.18
Hepatocyte nuclear factor 3 (HNF3), a critical hepatic transcriptional factor, serves a pivotal role in metabolism regulation and in the hepatic metabolism. In this report, we developed a method for reconstruction of damaged hepatic cells by applying the nanoparticle-delivery system. We developed a method to efficiently deliver a hepatic-specific factor that can improve cell therapy and tissue engineering in the treatment in an AHF model. We herein investigated the hepatoprotective function of novel nanoparticles (NNPs) in a carbon tetrachloride (CCl4), a severe toxic substance causing liver damage, induced AHF mouse platform. Our findings revealed that NNPs could effectively protect the injured hepatic organs and significantly improve the outcomes in mice pretreated with CCl4. In this study, we demonstrated that this novel NNP system would provide a new way for medical research and develop a drug-driven strategy for recovering hepatic disorders.
2.1. Synthesis of NNPs
The study materials, biosynthetic products, and chemicals were delivered from commercial companies. NNPs were obtained and synthesized by modifying N-trimethoxysilylpropyl-N,N,N-trimethylammonium chloride (TA). All used chemicals were without further purification.
2.2. Real-time reverse transcription-polymerase chain reaction
In this study our protocols are as follows: the RNA was isolated from cells using Trizol reagent and the QiagenRNAeasy (Qiagen, USA) column was used for purification, according to the manufacturer’s instructions. The RNA was extracted and quantized by using UltroSpec 3100 Pro, and 1 μg of RNA was reversely transcribed with a SuperScript III reverse transcriptase kit (Invitrogen, USA). All cDNA amplification was done in a total volume of 15 mL containing 0.5 mM of each primer, 6 mM MgCl2, 3 mL LightCycler FastStart DNA Master SYBR green I (Roche Diagnostics, Pleasanton, CA), and 1.5 mL of 1:12 diluted cDNA, and then evaluated by using LightCycler Roche Diagnostics. GAPDH housekeeping gene was amplified as a reference standard in each experiment. Moreover, the final steps, including PCR-reactions, were done in triplicate and then heated to 95°C for 10 minutes, as done by 35 cycles of denaturation at 95°C for 10 seconds, and further annealing at 60°C for 5 seconds, and the extension of final product at 72°C for 20 seconds.
2.3. Cell viability assay
HepG2 and PLC were maintained in Dulbecco’s modified Eagle’s medium (DMEM) (Invitrogen, Carlsbad, CA). The cell viability and proliferation ability of HepG2 and PLC was measured by MTT test and other assays. In brief, NNPs were seeded on 24-well plates at a density of 2 × 104 cells/well in medium and submitted to MTT assay (Sigma-Aldrich). Cells were incubated with 0.25 g/L MTT for 4 hours at 37°C, and added with 100% isopropanol to terminate the reaction. The MTT formazan product was measured by using a microplate reader and the optical density (OD) at 570 nm were detected (SpectraMax 250, Molecular Devices, CA, USA). The cell viability was measured using the OD values.
2.4. Animal model of liver injury
The animal study has been approved by TVGH Animal Committee, and the principles of Laboratory Animal Care were performed. Eight-weeks-old mice (25 to 30 g, male B6 mice) were used for our experiments. Intraperitoneal injection of CCl4 (2.5 mL/kg body weight) was given to induce fulminant hepatic failure. Six hours after the administration of CCl4, B6 mice were intraperitoneally injected with PBS, NP, or NP-HNF3 at four different dosages. After the administration of CCl4, the evaluation was performed at 24, 48, or 72 hours in recipients of PBS, NP, or NP-HNF3 to monitor hepatic damage.
2.5. Determination of intracellular reactive oxygen species production and nitrate/nitrite concentration
Cells were incubated with 5 μmol/L of 2′,7′-dichlorofluorescein diacetate (DCFH-DA) in a culture medium for 30 minutes at 37°C, and then flow cytometry analysis was performed to measure intracellular reactive oxygen species (ROS) production by the probe DCFH-DA (Molecular Probes, Eugene, USA). Total nitrite in liver tissue was assayed by adding 100 μL Griess reagent (0.05% naphthalethylenediamine dihydrochloride and 0.5% sulphanilamide in 2.5% phosphoric acid) to each sample. The total nitrite/nitrate concentration of the sample was measured at OD 550 nm in comparison with a standard solution of sodium nitrate prepared in saline.
2.6. Malondialdehyde assay
The measurement of malondialdehyde (MDA) in liver tissue was performed by using an enzyme-linked immunosorbent assay (ELISA) reader (Bio-Rad Laboratories, Hercules, CA, USA) by determining absorbance at 530 nm.
2.7. Statistical analysis
In this study, we used the statistical analysis as mean ± SD. Statistical analysis was performed using Student’s t test or a one-way or two-way analysis of variance test followed by Turkey’s test, as appropriate. p < 0.05 was considered to be statistically significant.
3.1. Characterization and preparation of NNPs
In this study, we attempted to develop NNPs for the efficient gene delivery of HNF3 and examined NNP-based delivery of HNF3 could rescue acute severe hepatic failure. The uniform nanoparticles were prepared under proper ratio. Briefly, tetraethyl orthosilicate and cetyltrimethylammonium bromide (surfactant) were prepared and used in this study. The NNPs were characterized by FT-IR and 1H NMR (400 MHz, DMSOd6, ppm; data not shown) spectroscopies. NNP-sbPEI was synthesized using the aminolysis reaction of polyurethane (c) and small branch polyethyleimine (MW = 600) (sbPEI). The average size of NNPs is 100 nm. The ultramicroscopic structure was shown in Figure 1A, B.
3.2. NNP-mediated delivery of HNF3 into hepatoma cells
In this study, we used the NNPs, a noncytotoxic vehicle, which are capable of high transfection activity in a delivery vehicle. After the generation and characterization of NNPs, stable HNF3-overexpressing hepatic cells were quickly generated using the NNP-delivery system (Fig. 1C) with plasmid vectors. The pHNF3 was loaded on NNPs complexes by electrostatic adsorption. We further explored the optimal concentration and mixture ratio for HNF3cDNA with the NNPs. Notably, our results demonstrated that the ratio of pHNF3β to NNP is fixed on 1:32 for the gene-delivery studies. To further measure the roles of NNP-HNF3 gene transduction in hepatic cells in vitro, an empty vector-delivery control and NNP-HNF3 was produced simultaneously and transfected into hepatic cell lines (Fig. 2A). The transfection of HNF3 into HepG2 cells is shown in Figure 2A. Moreover, our data showed that the CCl4-mediated toxicity could further damage the liver cell in vitro. Importantly, the results of cell proliferation and viability assay showed that the treatment of NNP-HNF3 may protect the CCl4-damaged hepatic cell (HepG2 and PLC cells in Fig. 2B).
3.3. Amelioration of fulminant hepatic failure by transplantation of NNP-HNF3-treated hepatocytes
Furthermore, we examined whether NPP-HNF3 could improve acute liver damage and hepatic failure in CCl4-treated mouse with acute liver injury. To detect acute injuries, a dosage of CCl4-2.5 mL/kg was administrated intraperitoneally. B6 mice were delivered into recipients with either PBS or NNP. The effect of NNP-HNF3 was not obvious among all given NNPs with or without HNF3 plasmids, such as 2 × 105, 5 × 105, 2 × 106, and 5 × 106/kg. NNP alone showed no additional effect on the cell therapy, whereas transplantation of NNP-HNF3 in CCl4-treated mice led to a remarkable reduction in necrotic area in CCl4-induced AHF (Fig. 3A). We next investigated that the treatment by injecting NNP-HNF3 in CCl4-treated mice reduced the production of oxidative stress in vivo. MDA and nitrate/nitrite are indicative of oxidative damage. Furthermore, using the toxic model such as CCl4 administration elicited the production of MDA, nitrate/nitrite, and ROS in livers from all treated-groups in CCl4-treated mice (Figure 3B–D). The CCl4-induced production of MDA, nitrate/nitrite, and ROS were not affected by NNP-HNF3 transplantation (Figure 3B–D). Moreover, the CCl4-induced production of MDA, nitrate/nitrite, and ROS significantly reduced in livers from recipients of NNP-HNF3-based transplantation. N-acetyl-cysteine (NAC), an antioxidant, can minimize ROS. Administration of NAC decreased the CCl4-induced damages in causing the oxidative ROS and toxicity in the different groups with NNP-HNF3 or control treatment. The maximal inhibition of oxidative substances was observed in recipients of NNP-HNF3-based therapeutics. In addition, the mobility and capabilities of motor activity studies are done. After treated with CCl4 for seventy-two hours, the result showed that the total mobility and ambulatory movements could be significantly increased and improved in groups of NNP-HNF3, compared to all other groups (Fig. 4).
Herein we discovered that NNP-HNF3 exhibited a prominent antioxidant system both in vitro and in vivo. For ensuring the hepatoprotective property of NNPs in vivo, we tested the cytoprotection of NNPs after cell transplantation into the mouse model of AHF, induced by the hepatotoxic CCl4. Importantly, the gene delivery of NNPs-HNF3 effectively rescued these CCl4-treated mice. Our key results further presented to significantly decrease liver damage area and oxidative substances with recovering hepatic functions and mobility activities.
Previous studies have showed that hepatic encephalopathy causes high mortality rate, occurrence of confusion, as well as changing level of consciousness and coma.20–23 This is the major complication associated with fulminant hepatic failure. This study showed that NNP could effectively improve AHF. Using this NNP gene-delivery system, we can provide a potential platform of NNP-HNF3 presenting therapeutic effect against hepatic encephalopathy. Our findings have showed the evidence to demonstrate NNP-HNF3 treatment could not only protect liver damage but also effectively improve toxic-induced hepatic encephalopathy.
In conclusions our findings showed that NNPs effectively rescued CCl4-induced AHF, and the hepatoprotective effect of NNP-HNF3 could present the potential roles in modulating hepatoprotective effects to repair injured tissues. In the future, it will be a potential platform to take the advantage of the hepatic protection function and efficacy of the NNP-HNF3 in fulminant liver failure. In addition, the NNP system carrying the gene products, small molecules, and pharmaceutic peptides may serve as an alternative to therapeutics to treat hepatic failure.
This study was supported by research grants from the Ministry of Science and Technology (MOST 103-2314-B-075-027-MY3), Taipei Veterans General Hospital (V107C-145), the Joint Projects of National Yang-Ming University and Cheng-Hsin General Hospital, and the Joint Projects of National Yang-Ming University and Far-Eastern Memorial Hospital from 2015 to 2018, Taiwan.
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