Liver Targeting Research Articles

Mulberry anthocyanin extract ameliorates insulin resistance by regulating PI3K/AKT pathway in HepG2 cells and db/db mice

This study evaluated the capacity of mulberry anthocyanin extract (MAE) on insulin resistance amelioration in HepG2 cells induced by high glucose and palmitic acid and diabetes-related metabolic changes in type 2 diabetic mice. In vitro, MAE alleviated insulin resistance in HepG2 cells and increased glucose consumption, glucose uptake and glycogen content. Enzyme activities of phosphoenolpyruvate carboxykinase (PEPCK) and glucose-6-phosphatase (G6Pase) were decreased due to PPARγ coactivator 1α (PGC-1α) and forkhead box protein O1 (FOXO1) inhibition. Furthermore, phosphorylation of protein kinase B (AKT) and glycogen synthase kinase-3β (GSK3β) in model cells was recovered after treated with MAE, leading to an up-regulation of glycogen synthase 2 (GYS2), and this effect was blocked by the phosphatidylinositol-3-kinase (PI3K) inhibitor LY294002. In vivo, MAE supplementation (50 and 125 mg/kg body weight per day) markedly decreased fasting blood glucose, serum insulin, leptin, triglyceride and cholesterol levels and increased adiponectin levels in db/db mice. The improvement of related metabolic parameters was in part associated with the impact of MAE on activating AKT and downstream targets in liver, skeletal muscle and adipose tissues. In summary, these findings suggest that MAEs have potential benefits on improving dysfunction in diabetic mice and mitigating insulin resistance in HepG2 cells via activation of PI3K/AKT pathways.

Abstract 2055: A first-in-class hemoglobin-floxuridine conjugate for the treatment of advanced stage liver cancer

Abstract Hepatocellular carcinoma (HCC) is the second most common cause of death from cancer worldwide; few treatment options are available, especially for advanced stage disease. Therapure has developed a novel drug delivery platform based upon the attachment of drugs to hemoglobin (Hb) as a means of targeting the liver. TBI 302 is a hemoglobin-drug conjugate (HDC) designed to deliver the anticancer drug floxuridine to the liver to improve treatment for HCC. Systemic toxicity associated with free floxuridine restricts its use to locoregional administration (0.2-0.5 mg/kg/d) via continuous hepatic arterial infusion. Although hepatic arterial infusion of floxuridine can reduce hepatic tumor burden, toxicity from floxuridine and complications associated with direct hepatic infusion pumps can be significant. HDC technology exploits the natural pathway for hemoglobin clearance through the liver to provide selective drug targeting while preserving floxuridine activity following standard intravenous (IV) infusion. In a preclinical efficacy study of TBI 302, mice bearing human liver cancer cell line-derived orthotopic liver tumors received twice-weekly tail vein dosing of saline, 3.7 mg/kg floxuridine, or TBI 302. Following 6 weeks of treatment, 70% of the orthotopically implanted mice treated with 170.5 mg/kg TBI 302 (3.7 mg/kg floxuridine) had no measurable liver tumors, indicating suppression of tumor growth. In contrast, only 30% of mice treated with an equivalent dose of unconjugated floxuridine and 20% of mice treated with saline experienced liver tumor growth suppression. These results demonstrate the capability of the HDC platform to enhance the efficacy of cytotoxics through liver targeting. To establish a safe starting dose for a first-in-human Phase 1 trial, a GLP-compliant repeat dose preclinical safety study of TBI 302 in cynomolgus monkeys was conducted. TBI 302 administered by 1-hour IV infusion once per week for 8 weeks at doses of 2, 5, and 10.5 mg/kg (0.08, 0.19, and 0.4 mg/kg floxuridine, respectively) was well tolerated at all dose levels. Increasing doses of TBI 302 resulted in proportional increases in area under the concentration-time curve (AUC) and maximum concentration (Cmax) of total plasma floxuridine. No clinical signs or biochemical toxicity was associated with IV infusion of TBI 302. The no-observed-adverse effect level (NOAEL) of TBI 302 was determined to be the highest dose level of 10.5 mg/kg (0.4 mg/kg floxuridine). A Phase I safety study of TBI 302 as second-line therapy in HCC has been approved by the FDA. The primary objective is to determine safety and tolerability of TBI 302. Secondary objectives are to determine TBI 302 pharmacokinetics and effects on tumor burden. Therapure's HDC platform represents a new class of therapy that offers liver-specific targeting, while potentially reducing extra-hepatic toxicity of drugs. Citation Format: Steve Brookes, Murray J. Cutler, Jin Seog Seo, Gord Adamson, David Bell. A first-in-class hemoglobin-floxuridine conjugate for the treatment of advanced stage liver cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2055.

PH-sensitive pHLIP® coated niosomes.

Nanomedicine is becoming very popular over conventional methods due to the ability to tune physico-chemical properties of nano vectors, which are used for encapsulation of therapeutic and diagnostic agents. However, the success of nanomedicine primarily relies on how specifically and efficiently nanocarriers can target pathological sites to minimize undesirable side effects and enhance therapeutic efficacy. Here, we introduce a novel class of targeted nano drug delivery system, which can be used as an effective nano-theranostic for cancer. We formulated pH-sensitive niosomes (80-90 nm in diameter) using nonionic surfactants Span20 (43-45 mol%), cholesterol (50 mol%) and 5 mol% of pH (Low) insertion peptide (pHLIP) conjugated with DSPE lipids (DSPE-pHLIP) or hydrophobic fluorescent dye, pyrene, (Pyr-pHLIP). In coating of niosomes, pHLIP was used as an acidity sensitive targeting moiety. We have demonstrated that pHLIP coated niosomes sense the extracellular acidity of cancerous cells. Intravenous injection of fluorescently labeled (R18) pHLIP-coated niosomes into mice bearing tumors showed significant accumulation in tumors with minimal targeting of kidney, liver and muscles. Tumor-targeting niosomes coated with pHLIP exhibited 2-3 times higher tumor uptake compared to the non-targeted niosomes coated with PEG polymer. Long circulation time and uniform bio-distribution throughout the entire tumor make pHLIP-coated niosomes to be an attractive novel delivery system.

Optimization of Transforming Growth Factor-β1 siRNA Loaded Chitosan-Tripolyphosphate Nanoparticles for the Treatment of Colorectal Cancer Hepatic Metastasis in a Mouse Model.

Metastatic liver disease is the most frequent complication of colorectal cancer (CRC), and the development of liver-targeted nanoparticles for drug delivery is a promising therapeutic approach. However, to improve the efficacy of passive drug delivery, its release rate at the sites of liver metastases should be maximized while minimizing drug uptake in nontargeted cells. Herein, we report the development and use of tripolyphosphate (TPP) modified chitosan (CS) nanoparticles loaded with small interfering RNA (siRNA) directed against transforming growth factor β1 (TGF-β1), which promotes tumorigenesis in advanced CRC. The nanoparticles efficiently inhibited CRC hepatic metastasis in an animal model. Particles of 300 nm in size and zeta potential at 20 mV showed a more striking liver-targeting effect. A weight ratio of CS/TPP of 8:1 for particles with TGF- β1 siRNA loaded at a concentration of 20 μM at pH 7.5 showed good pH-responsive drug release when exposed to a CRC homogenate at pH 6.5. In vivo, CS-TPP/TGF- β1 siRNA nanoparticles significantly reduced the volume and number of CRC metastatic foci. This was accompanied by the downregulation of TGF- β1 expression in the tumor microenvironment, inhibition of tumor associated macrophage formation, and improvement of the immune microenvironment. These results indicate that it is possible to achieve effective passive liver targeting by optimizing the processing parameters. The design of nanoparticles carrying siRNA against overexpressed oncogenes provides an excellent platform for the development of an efficient liver cancer therapy.

Feasibility study on image guided patient positioning for stereotactic body radiation therapy of liver malignancies guided by liver motion.

BackgroundFiducial markers are the superior method to compensate for interfractional motion in liver SBRT. However this method is invasive and thereby limits its application range. In this retrospective study, the compensation method for the interfractional motion using fiducial markers (gold standard) was compared to a new non-invasive approach, which does rely on the organ motion of the liver and the relative tumor position within this volume.MethodsWe analyzed six patients (3 m, 3f) treated with SBRT in 2014. After fiducial marker implantation, all patients received a treatment CT (free breathing, without abdominal compression) and a 4D-CT (consisting of 10 respiratory phases). For all patients the gross tumor volumes (GTVs), internal target volume (ITV), planning target volume (PTV), internal marker target volumes (IMTVs) and the internal liver target volume (ILTV) were delineated based on the CT and 4D-CT images. CBCT imaging was used for the standard treatment setup based on the fiducial markers. According to the patient coordinates the 3 translational compensation values (tx, ty, tz) for the interfractional motion were calculated by matching the blurred fiducial markers with the corresponding IMTV structures. 4 observers were requested to recalculate the translational compensation values for each CBCT (31) based on the ILTV structures. The differences of the translational compensation values between the IMTV and ILTV approach were analyzed.ResultsThe magnitude of the mean absolute 3D registration error with regard to the gold standard overall patients and observers was 0.50 cm ± 0.28 cm. Individual registration errors up to 1.3 cm were observed. There was no significant overall linear correlation between the respiratory motion and the registration error of the ILTV approach.ConclusionsTwo different methods to calculate the translational compensation values for interfractional motion in stereotactic liver therapy were evaluated. The registration accuracy of the ILTV approach is mainly limited by the non-rigid behavior of the liver and the individual registration experience of the observer. The ILTV approach lacks the accuracy that would be desired for stereotactic radiotherapy of the liver.

Non-alcoholic fatty liver and the gut microbiota

BackgroundNon-alcoholic fatty liver (NAFLD) is a common, multi-factorial, and poorly understood liver disease whose incidence is globally rising. NAFLD is generally asymptomatic and associated with other manifestations of the metabolic syndrome. Yet, up to 25% of NAFLD patients develop a progressive inflammatory liver disease termed non-alcoholic steatohepatitis (NASH) that may progress towards cirrhosis, hepatocellular carcinoma, and the need for liver transplantation.In recent years, several lines of evidence suggest that the gut microbiome represents a significant environmental factor contributing to NAFLD development and its progression into NASH. Suggested microbiome-associated mechanisms contributing to NAFLD and NASH include dysbiosis-induced deregulation of the gut endothelial barrier function, which facilitates systemic bacterial translocation, and intestinal and hepatic inflammation. Furthermore, increased microbiome-modulated metabolites such as lipopolysaccharides, short chain fatty acids (SCFAs), bile acids, and ethanol, may affect liver pathology through multiple direct and indirect mechanisms. Scope of reviewHerein, we discuss the associations, mechanisms, and clinical implications of the microbiome's contribution to NAFLD and NASH. Understanding these contributions to the development of fatty liver pathogenesis and its clinical course may serve as a basis for development of therapeutic microbiome-targeting approaches for treatment and prevention of NAFLD and NASH. Major conclusionsIntestinal host–microbiome interactions play diverse roles in the pathogenesis and progression of NAFLD and NASH. Elucidation of the mechanisms driving these microbial effects on the pathogenesis of NAFLD and NASH may enable to identify new diagnostic and therapeutic targets of these common metabolic liver diseases.This article is part of a special issue on microbiota.

Zinc alpha2 glycoprotein alleviates palmitic acid-induced intracellular lipid accumulation in hepatocytes

Zinc alpha2 glycoprotein (ZAG) plays an important role in stimulating fat mobilization and lipolysis in adipose tissue, but its role in hepatic lipid metabolism remains unclear. Palmitic acid (PA) was used to stimulate HepG2 cells with ZAG overexpression or ZAG knock down (shRNA). Overexpression of ZAG significantly inhibited lipogenesis, promoted lipolysis and fatty acid β-oxidation, and attenuated PA-induced intracellular fat accumulation. Moreover, ZAG overexpression dramatically stimulated adiponectin expression in HepG2 cells. In contrast, knockdown of ZAG notably inhibited fatty acid β-oxidation, increased lipogenesis and lipid accumulation. Collectively, these data suggest that ZAG has the potential to alleviate hepatosteatosis, making it a promising therapeutic target for fatty liver.

SU‐F‐J‐89: Assessment of Delivered Dose in Understanding HCC Tumor Progression Following SBRT

Purpose:It is well documented that the delivered dose to patients undergoing radiotherapy (RT) is often different from the planned dose due to geometric variability and uncertainties in patient positioning. Recent work suggests that accumulated dose to the GTV is a better predictor of progression compared to the minimum planned dose to the PTV. The purpose of this study is to evaluate if deviations from the planned dose can contributed to tumor progression.Methods:From 2010 to 2014 an in‐house Phase II clinical trial of adaptive stereotactic body RT was completed. Of the 90 patients enrolled, 7 patients had a local recurrence defined on contrast enhanced CT or MR imaging 3–21 months after completion of RT. Retrospective dose accumulation was performed using a biomechanical model‐based deformable image registration algorithm (DIR) to accumulate the dose based on the kV CBCT acquired prior to each fraction for soft tissue alignment of the patient. The DIR algorithm was previously validated for geometric accuracy in the liver (target registration error = 2.0 mm) and dose accumulation in a homogeneous image, similar to a liver CBCT (gamma index = 91%). Following dose accumulation, the minimum dose to 0.5 cc of the GTV was compared between the planned and accumulated dose. Work is ongoing to evaluate the tumor control probability based on the planned and accumulated dose.Results:DIR and dose accumulation was performed on all fractions for 6 patients with local recurrence. The difference in minimum dose to 0.5 cc of the GTV ranged from −0.3–2.3 Gy over 3–5 fractions. One patient had a potentially significant difference in minimum dose of 2.3 Gy.Conclusion:Dose accumulation can reveal tumor underdosage, improving our ability to understand recurrence and tumor progression patterns, and could aid in adaptive re‐planning during therapy to correct for this.This work was supported in part by NIH P01CA059827.

Quercetin-loaded poly (lactic-co-glycolic acid)-d-α-tocopheryl polyethylene glycol 1000 succinate nanoparticles for the targeted treatment of liver cancer

Utilization of quercetin (QT) in clinics is limited by its instability and poor solubility. To overcome these disadvantages, we prepared QT as QT-loaded PLGA-TPGS nanoparticles (QPTN) and examined its properties and therapeutic efficacy for liver cancer. QT-loaded PLGA nanoparticles (QPN) and QT/coumarin-6-loaded PLGA-TPGS nanoparticles (QCPTN) with coumarin-6 as a fluorescent marker were also prepared to investigate the cellular uptake by HepG2 and HCa-F cells using a confocal laser scanning microscope (CLSM), and their effects on apoptosis of HepG2 cells were assessed with flow cytometry. The results measured using transmission electron microscopy, scanning electron microscopy and size analyses indicated that QPTN were stably dispersed sphere with diameter in the range of 100-200 nm. It indicated that the QT loading and encapsulation efficiency in QPTN reached 21.63% and 93.74%, respectively, and the accumulative drug release of QPTN was 85.8%, the QCPTN uptake in HCa-F and HepG2 cells were 50.87% and 61.09% using HPLC analysis, respectively. The results determined using an Annexin-PI flow cytometry indicated that QPTN could induce HepG2 cell apoptosis in a dose dependent manner. The results of histological examination and HPLC analysis confirmed that QPTN was targeted to liver cells. In vivo analysis using solid tumor-bearing mouse model indicated that QPTN could suppress tumor growth by 59.07%. Moreover, all the studied properties of QPTN were more desirable than those of QT-loaded PLGA nanoparticles (QPN). In conclusion, QPTN could be used as a potential intravenous dosage form for the treatment of liver cancer owing to the enhanced pharmacological effects of QT with increased liver targeting.

169. Expression Studies of Ornithine Transcarbamylase in Liver Using Minicircle Vectors for Gene Therapy: Flag-Tagging of a Mitochondrial Enzyme and Stable Expression Under Control of an Endogenous Otc Promoter-Enhancer

Ornithine transcarbamylase deficiency (OTCD) is the most common inherited defect of the urea cycle resulting in severe hyperammonemia and death if left untreated. We are aiming at correcting OTCD using naked-DNA minicircle (MC) vector mediated gene therapy in the spfash mouse model with low residual OTC activity. A critical parameter is delivery to periportal hepatocytes where the urea cycle is located because of metabolic zonation in the liver. To distinguish between MC-born and endogenous OTC enzyme, we generated an expression cassette with an internally Flag-tagged OTC enzyme. Internal epitope tagging is required because of the N-terminal mitochondrial import sequence. A corresponding Flag-tag sequence was introduced C-terminally of the mitochondrial import signal. The tagged protein performed similar to its non-tagged OTC enzyme upon hydrodynamic tail vein injection for liver targeting in spfash mice, indicating that the tag neither interferes with the mitochondrial import nor the formation of a functional OTC trimer. Furthermore we generated an endogenous Otc promoter-enhancer construct, termed PmO1, for potential specific or “natural” OTC transgene expression. According to others, the promoter (672 bp) sequence derived from mouse Otc was not sufficient for liver specificity (Veres et al, J Biol Chem 261: 7588-7591, 1986) and requires a corresponding enhancer sequence (Nishiyori et al, J Biol Chem 269: 1323-1331, 1994). An enhancer sequence (232 bp) originating from rat Otc was used that contains several binding sites for liver-selective transcription factors (Murakami et al, Mol Cell Biol 10: 1180-1191, 1990). The resulting MC-vector expressing OTC from this PmO1 promoter-enhancer construct was delivered to spfash mice via hydrodynamic tail vein injection, resulting in similar enzymatic activity in whole liver extracts compared to wild-type mice. Vectors designed to express Flag-tagged OTC driven from a natural Otc promoter might be useful to study biodistribution and/or long-term stable expression after MC-based gene therapy for OTCD.

595. Enhanced Liver Transduction and Efficient Protection from Pre-Existing Neutralizing Antibodies with Exosome-Associated AAV8 Vectors

Adeno-associated virus (AAV) based vectors are ideal tools for in vivo gene transfer. The excellent safety profile and the highly efficient targeting of hepatocytes in vivo have been demonstrated in both preclinical studies and clinical trials. Although AAV vectors are not strongly immunogenic, they can give rise to both cellular and humoral immune responses. Additionally, as a result of exposure to wild-type AAV, an important fraction of humans harbor pre-existing neutralizing antibodies against the viral capsid, which can prevent successful transduction by AAV vectors. This pose a serious obstacle to the widespread use of AAV vectors in gene therapy. Recently, we have shown that AAV can associate with exosomes (exo-AAV) ultracentrifuged media samples from 293T producer cells. Exosome-associated AAV1, 2, and 9 serotype capsids were tested and found to have enhanced transduction and antibody evasion capabilities compared to conventional AAV vectors. With the goal of developing an enhanced vector for liver directed gene therapy, we sought to characterize the in vivo transduction and biodistribution profile of exo-AAV8 compared to conventional cell-lysated harvested, iodixanol-gradient purified AAV8 vectors purified from the same preparation. The efficiency of liver targeting of conventional AAV8 vs. exo-AAV8 expressing human factor IX (F.IX) under the control of liver specific promotor were tested in naive and pre-immunized animals. C57BL/6 mice (n=5/group) were passively immunized with intravenous human immunoglobulin (IVIg) intraperitoneally, followed 24h later by the intravenous administration of either conventional AAV8 or exo-AAV8 vectors expressing coagulation factor IX (F.IX, 5×1010 vg/mouse). exo-AAV8 completely shielded the capsid vector from neutralizing antibodies at IVIg doses between 0.5mg and 2mg/mouse (NAb titer ~ 1:3.16), resulting in equivalent levels of F.IX transgene expression to naive animals treated with conventional AAV8 vector. At the highest IVIg dose tested (8mg/mouse), residual levels of F.IX transgene expression were about 30% of naive animals treated with conventional AAV8 vectors. We next evaluated the efficiency of exo-AAV8 vs. AAV8 vectors in naive animals. In male mice, no statistically significant difference in F.IX transgene expression levels was observed between the two vector types; also indicated by the similar pattern of vector genome biodistribution. Interestingly, female mice (in which the efficiency of liver transduction with AAV is extremely low compared with male animals) treated with exo-AAV8 showed a dramatic increase in transgene expression, comparable to that of male mice receiving conventional AAV8 vectors. In conclusion, exo-AAV8 vectors present an enhanced liver transduction profile compared with conventionally purified AAV vectors, both in naive female animals and in animals carrying anti-capsid neutralizing antibodies.

In vitro Toxicity Evaluation and in vivo Biodistribution of Polymeric Micelles Derived from Poly(ethylene glycol)-b-poly(benzyl malate) Copolyme

Background: The development of polymeric micelles for site-specific drug delivery is an exponentially growing field of research. In this context, we have designed two degradable amphiphilic copolymers, the poly(ethylene glycol)-b-poly(benzyl malate) (PEG42-b-PMLABe73) and the biotin-poly(ethylene glycol)- b-poly(benzyl malate) (Biot-PEG62-b-PMLABe73). The copolymer bearing biotin residue was synthesized in order to formulate micelles for grafting biotinylated cyclic RGD peptide onto their surface via the bridging streptavidin. Objective: Our study aimed at investigating the in vitro and in vivo toxicity of such micelles and to evaluate the potential of these nanovectors for hepatocyte-targeted drug delivery. Methods: The toxicity of micelles obtained by the nanoprecipitation method and characterized by dynamic light scattering and zeta potential measurement was evaluated in vitro using the differentiated hepatocyte-like HepaRG cells and in vivo in mice. Results: The micelles derived from PEG42-b-PMLABe73, Biot-PEG62-b-PMLABe73 and RGDBiot-Strept- Biot-PEG62-b-PMLABe73 did not affect the cell proliferation and apoptotic indexes in HepaRG hepatoma cells and were well tolerated in mice following systemic injection. The hydrophobic fluorescent probes DiD oil and DiR were efficiently encapsulated in PEG-b-PMLABe-derived micelles allowing the visualization of their uptake into HepaRG cells. Furthermore, the addition of RGD peptide onto micelles strongly enhanced the cell uptake in vitro and liver targeting in vivo. Conclusion: These data demonstrate the low toxicity of poly(benzyl malate) derived copolymers towards hepatocyte- like cells and emphasize their potential use for the design of liver targeting nanovectors

Highly biocompatible amphiphilic perylenediimide derivative for bioimaging

We report the synthesis and biological studies of a fluorescence dye with an oligoethylene glycol substituted (OEG) perylene centered dye N,N’-(2,6-diisopropylphenyl)-1-[oligo(ethylene glycol)methyl ether]-1,6,7,12-trichloroperylene-3,4:9,10-tetracarboxdiimide (PDI-OEG). The activity of the dye is juxtaposed with a precursor molecule without the OEG substitution. The OEG substitution contributes to the increased biocompatibility of PDI-OEG. Cell viability studies lead to the survival of more than 80% of the PDI-OEG cultured cells endorsing its biocompatibility. Fluorescence imaging studies were carried out using multiple cell lines. Ex-vivo studies involving nude mice were used to establish liver and lung specific organ targeting of PDI-OEG. This fluorophore is an excellent example of a stable and biocompatible red emitting small molecule for bioimaging.

EP-1749: Real-time 4D ultrasound tracking of liver and kidney targets for external-beam radiotherapy

EP-1749: Real-time 4D ultrasound tracking of liver and kidney targets for external-beam radiotherapy

Severe Hepatitis Promotes Hepatocellular Carcinoma Recurrence via NF-κB Pathway-Mediated Epithelial-Mesenchymal Transition after Resection.

Surgical resection is considered as a curative treatment modality for hepatocellular carcinoma; however, the incidence of postoperative tumor recurrence is high, leading to worse patient survival. Persistent hepatitis (inflammation) is one of the risk factors of tumor recurrence after surgical resection. The aim of this study is to investigate the underlying mechanisms linking liver inflammation to hepatocellular carcinoma progression. In this study, we used a cytokine array to identify important cytokines whose levels are increased in liver microenvironment with severe hepatitis. We evaluated the morphologic changes, migration and invasion ability, and signal transduction in hepatocellular carcinoma cells with or without inflammatory cytokine in vitro Finally, we analyzed the NF-κB signal pathway in tumor specimens from 232 patients with hepatocellular carcinoma by immunohistochemical staining. The proinflammatory cytokine TNFα was increased in the peritumoral microenvironment and contributed to tumor recurrence and metastasis. Specifically, TNFα promoted hepatocellular carcinoma cancer cell migration, invasion, and epithelial-mesenchymal transition (EMT) by upregulating the transcriptional regulator, Snail. We identified Snail as a direct target gene downstream of the TNFα-mediated canonical NF-κB activation. In addition, tumor recurrence-free survival of hepatocellular carcinoma patients correlated negatively with high p65 and Snail expression and positively with high E-cadherin expression. Our results establish a signaling axis that explains how inflammatory tumor microenvironment promotes hepatocellular carcinoma recurrence and metastasis. These findings suggest that controlling liver inflammation and/or targeting NF-κB-mediated Snail expression may be a potential therapeutic strategy to prevent hepatocellular carcinoma recurrence after hepatectomy.

Dextran based nanosized carrier for the controlled and targeted delivery of curcumin to liver cancer cells

Curcumin (Cur), a poly phenolic yellow colored compound present in Indian spice turmeric, has a wide variety of biological properties. Bioavailability of Cur is limited by its low water solubility, rapid metabolism and low stability. In the present study, we mainly focus on synthesis and characterization of dextran based nano-sized drug carrier (GHDx) for the delivery of Cur. A liver targeting moiety is incorporated in GHDx so as to improve the therapeutic efficiency and decrease adverse effects of conventional cancer therapy. The effect of different parameters on grafting variables was studied. GHDx was characterised by FTIR, 1H NMR XRD, TG/DTG, TEM, SEM, AFM, DLS and zeta potential analyses. Adsorption experiments were carried out for drug loading. Swelling of GHDx was studied as a function of pH and temperature. Three step release of Cur from GHDx was confirmed by analyzing in vitro release data in simulated intracellular pH using different kinetic models. In vitro cytotoxicity analysis on L929 and Hep G2 cells shows that GHDx is safe carrier while Cur loaded GHDx exhibits high toxicity with slow drug release towards hepatic cells. The results show that the GHDx can be customized as a stimuli sensitive potential carrier for the delivery of drugs.

Soluble (pro)renin receptor via β-catenin enhances urine concentration capability as a target of liver X receptor

The extracellular domain of the (pro)renin receptor (PRR) is cleaved to produce a soluble (pro)renin receptor (sPRR) that is detected in biological fluid and elevated under certain pathological conditions. The present study was performed to define the antidiuretic action of sPRR and its potential interaction with liver X receptors (LXRs), which are known regulators of urine-concentrating capability. Water deprivation consistently elevated urinary sPRR excretion in mice and humans. A template-based algorithm for protein-protein interaction predicted the interaction between sPRR and frizzled-8 (FZD8), which subsequently was confirmed by coimmunoprecipitation. A recombinant histidine-tagged sPRR (sPRR-His) in the nanomolar range induced a remarkable increase in the abundance of renal aquaporin 2 (AQP2) protein in primary rat inner medullary collecting duct cells. The AQP2 up-regulation relied on sequential activation of FZD8-dependent β-catenin signaling and cAMP-PKA pathways. Inhibition of FZD8 or tankyrase in rats induced polyuria, polydipsia, and hyperosmotic urine. Administration of sPRR-His alleviated the symptoms of diabetes insipidus induced in mice by vasopressin 2 receptor antagonism. Administration of the LXR agonist TO901317 to C57/BL6 mice induced polyuria and suppressed renal AQP2 expression associated with reduced renal PRR expression and urinary sPRR excretion. Administration of sPRR-His reversed most of the effects of TO901317. In cultured collecting duct cells, TO901317 suppressed PRR protein expression, sPRR release, and PRR transcriptional activity. Overall we demonstrate, for the first time to our knowledge, that sPRR exerts antidiuretic action via FZD8-dependent stimulation of AQP2 expression and that inhibition of this pathway contributes to the pathogenesis of diabetes insipidus induced by LXR agonism.

The impact of retinol loading and surface charge on the hepatic delivery of lipid nanoparticles

The present work developed lipid nanoparticles to determine whether retinol loading and surface charge influenced liver targeting and biodistribution. Silibinin for treating liver fibrosis was used as the active model. The capability of nanoparticles to suppress hepatic stellate cells (HSCs) was investigated by examining cell viability and α-smooth muscle actin (α-SMA). The biodistribution of the nanocarriers in rats was monitored by real-time and organ bioimaging after an intravenous injection. Silibinin concentration in the organs was detected as well. Anionic nanoparticles showed a mean size of around 260nm, which was greater than that of cationic nanoparticles (about 170nm). The encapsulation percentage of silibinin was >98% for both anionic and cationic nanoparticles. All nanoparticles tested were able to be ingested into HSCs, with no difference between the formulations. The positive nanoparticles produced activated HSC apoptosis much more strongly than negative nanoparticles. The α-SMA suppression exhibited a contrary trend. The nanoparticles rapidly accumulated in the liver and spleen. Retinol incorporation in nanoparticles offers an active targeting approach to the liver via retinol binding protein (RBP). The negatively charged formulation containing retinol achieved higher uptake and longer retention in the liver than the other formulations. Silibinin inclusion in nanoparticles significantly decreased lung deposition and increased liver uptake. The lipid nanosystems promoted silibinin distribution to the liver by 2–3-fold compared to the free control. A better liver-specific selectivity was obtained by retinol-loaded anionic nanocarriers. It is important to optimize the formulations of the lipid nanoparticles for maximizing hepatic targeting.

Factors limiting the extent of absolute bioavailability of pradefovir in rat

1. Pradefovir was designed as an oral liver target prodrug of 9-(2-phosphonylmethoxyethyl) adenine (PMEA). Liver targeting arises through first pass hepatic metabolism by cytochrome P-450 3A4 (CYP3A4). For CYP3A4 primarily exists in intestines and liver, intestinal metabolism may impair its liver selectivity and oral bioavailability, and then impair its efficacy and safety. It was important to reveal details of the disposition of pradefovir in intestines and liver in a preclinical study.2. The absolute bioavailability of pradefovir was 4.75% based on the intravenous and oral AUC0–24 h in rats. Pradefovir was stable in intestinal segments and microsomes. The fractions of the dose absorbed from the GI tract were 20.3% and 15.3% from intravenous and oral administration of pradefovir in rats and portal vein-cannulated rat models, respectively. The liver extraction ratio was predicted to be 49.2% from liver microsomes system, based on the monitoring substrate loss rate. Rat intestines’ Ussing chamber experiment indicated that P-glycoprotein (P-gp) transporter and paracellular pathway may involve in intestinal transportation.3. Activation of pradefovir mainly occurs in the liver. Low intestinal absorption was the main reason of low bioavailability of pradefovir in rats. The result was suggestive for the disposition of pradefovir in human intestine and liver.

Highly specific electrochemical sensor of tumor marker using multi-nanoparticle labeling based 3D origami paper analytical device

and synthesis of a fluorescent off–on nanoprobe for MMP families (for an instance, collagenase IV, including MMP2, MMP4, and MMP9). The fabricated fluorescent nano-switch is the fluorescein isothiocyanate (FITC)-attached gold nanoparticle (GNP). The spacer inserted between FITC andGNP is a small peptide containing the core substrate sequence of MMPs and a free thiol group on the terminal cysteine. To ensure the desirable solubility, stability, and in vivo circulation of nano-switch, thiolated polyethylene glycol monomethyl ether (HS-mPEG2000) was also conjugated to the surface of GNP. The structure of the nano-switch was optimized through adjusting the molar ratio of GNP vs. p-FITC, investigating the effect on the addition of HS-mPEG2000, and evaluating fluorescence recovery efficiency of the nano-switch. Investigated using confocal laser fluorescence microscope technology, the cellular uptake result showed that with the help of GNPs, the nano-switch can bemore easily in taken byHepG2 cells (liver hepatocellular carcinoma) and lighten the greenfluorescence of FITC around GNPs inside cells. However, the green fluorescence was only found in the cell culture medium for the p-FITC group. Furthermore, tissue frozen section results demonstrated that (1) the nano-switch showed higher fluorescence intensity in tumor tissues; (2) the fluorescence recovery of the nano-switch was not only found in the margin but also in the center of tumor tissues; (3) the nano-switch exhibited lower distribution in normal tissues including heart, spleen, and lung; and (4) when comparing with p-FITC, the similar and higher accumulation of the nano-switch in liver and kidney, respectively, indicated its liver targeting and renal elimination characters.