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[Display omitted] Asialoglycoprotein receptors (ASGPRs) are highly expressed on hepatocytes and have been used for liver-targeted delivery and hepatocellular carcinoma (HCC) therapy. However, targeted delivery of bortezomib (BTZ) to HCC has not been reported. In this study, N-stearyl lactobionamide (N-SALB) with galactose (Gal) moiety was synthesized as a targeting agent and its structure was confirmed by FT-IR and NMR analyses. N-SALB surface-modified solid lipid nanoparticles (SLNs) loaded with BTZ (Gal-SLNs/BTZ) were developed to target BTZ delivery into HCC cancer cells. The Gal-SLNs/BTZ had an average particle size of 116.3 nm, polydispersity index (PDI) of 0.210, and zeta potential of −13.8 mV. TEM analysis showed their nanometer-sized spherical morphology. The encapsulation efficiency (EE) and drug loading (DL) capacity were 84.5 % and 1.16 %, respectively. Release studies showed that BTZ loaded inside the SLNs was slowly released over a period of 72 h at pH 7.4. Flow cytometry analysis showed significantly higher intracellular uptake of N-SALB-targeted nanoparticles than non-targeted nanoparticles in HepG2 cells. All lipid formulations showed good biocompatibility in the cytotoxicity study using MTT assay. Concentration-dependent cytotoxicity was observed for all formulations, with N-SALB-targeted nanoparticles demonstrating more cytotoxicity against HepG2 cells. The highest percentage of apoptosis was obtained for N-SALB-targeted nanoparticles compared to non-targeted nanoparticles (42.2 % and 8.70 %, respectively). Finally, biodistribution studies in HepG2 bearing nude mice showed that the accumulation of targeted nanoparticles in the tumor was significantly higher than non-targeted nanoparticles. [ABSTRACT FROM AUTHOR] more...

A series of HepDirect pH responsive fluorescence probes of glycosyl-rhodamine are synthesized. The recognition trend of glucoside < lactoside < galactoside illuminates that the galactose is a targeting group toward HepG2 cells and its recognition is affected by the bulk of targeting group. The HepG2 cells binding trend of RDGal1< RDGal2< RDGal3 supports the cluster effect of galactose. RDGal3 with the optimum targeting potential is verified to selectively recognize HepG2 cells via a galactose dependent ASGPR binding mechanism. These nontoxic probes could be controllably activated by the acidic microenvironment of cancer cells, which is very significant to achieve diagnosis of hepatocellular carcinoma and provide a novel HepDirect carrier for precision treatment of hepatocarcinoma. [ABSTRACT FROM AUTHOR] more...

Reported here is the synthesis and biological evaluation of the asialoglycoprotein receptor (ASGP-R) targeted fourth generation poliamidoamine dendrimer (G(4)-PAMAM) loaded with sorafenib. The ASGP-R targeted dendrimer was obtained by conjugation of Lactobionic acid (La) to the G(4)-PAMAM dendrimer, followed by acetylation (Ac) of the free amino groups in order to reduce the non-specific interactions with the cell membrane. Moreover, by additionally grafting fluorescein (FITC), it was easy to characterize the internalization pathway and the intracellular fate of the targeted dendrimer Ac-La-G(4)-PAMAM-FITC. In vitro experiments performed on HepG-2 and HLE cell lines, allowed to study the ability of the dendrimers to affect the cell vitality. Confocal microscopy and cytofluorimetric analysis confirmed higher binding and uptake ability of the Ac-La-G(4)-PAMAM-FITC dendrimer in well differentiated and ASGP-R expressing human liver cancer cell line HepG-2 compared non-expressing HLE cells. Ac-La-G(4)-PAMAM-FITC dendrimer loaded with sorafenib was stable and showed sustained sorafenib release. As evidenced by the cytotoxicity studies, sorafenib included in the dendrimer maintained its effectiveness, and was able to produce a longer lasting effect over the time compared to molar equivalent doses of free sorafenib. This new targeted dendrimer appears to be a suitable carrier for the delivery of sorafenib to liver cancer cells expressing ASGP-R. [ABSTRACT FROM AUTHOR] more...

Adoptive immunotherapy leveraging chimeric antigen receptor-modified T (CAR-T) cells holds great promise for the treatment of cancer. However, tumor-associated antigens often have low expression levels in normal tissues, which can cause on-target, off-tumor toxicity. Recently, we reported that GPC3-targeted CAR-T cells could eradicate hepatocellular carcinoma (HCC) xenografts in mice. However, it remains unknown whether on-target, off-tumor toxicity can occur. Therefore, we proposed that dual-targeted CAR-T cells co-expressing glypican-3 (GPC3) and asialoglycoprotein receptor 1 (ASGR1) (a liver tissue-specific protein)-targeted CARs featuring CD3ζ and 28BB (containing both CD28 and 4-1BB signaling domains), respectively, may have reduced on-target, off-tumor toxicity. Our results demonstrated that dual-targeted CAR-T cells caused no cytotoxicity to ASGR1GPC3 tumor cells, but they exhibited a similar cytotoxicity against GPC3ASGR1 and GPC3ASGR1 HCC cells in vitro. We found that dual-targeted CAR-T cells showed significantly higher cytokine secretion, proliferation and antiapoptosis ability against tumor cells bearing both antigens than single-targeted CAR-T cells in vitro. Furthermore, the dual-targeted CAR-T cells displayed potent growth suppression activity on GPC3ASGR1 HCC tumor xenografts, while no obvious growth suppression was seen with single or double antigen-negative tumor xenografts. Additionally, the dual-targeted T cells exerted superior anticancer activity and persistence against single-targeted T cells in two GPC3ASGR1 HCC xenograft models. Together, T cells carrying two complementary CARs against GPC3 and ASGR1 may reduce the risk of on-target, off-tumor toxicity while maintaining relatively potent antitumor activities on GPC3ASGR1 HCC. [ABSTRACT FROM AUTHOR] more...

Liver cancer is one of the major diseases affecting human health. Modified drug delivery systems through the asialoglycoprotein receptor, which is highly expressed on the surface of hepatocytes, have become a research focus for the treatment of liver cancer. Resibufogenin (RBG) is a popular traditional Chinese medicine and natural anti-cancer drug that was isolated from Chansu, but its cardiotoxicity and hydrophobicity have limited its clinical applications. Galactosyl-succinyl-poloxamer 188 and galactosyl-succinyl-poloxamer 188-polylactide-co-glycolide (Gal-SP188–PLGA) were synthesized using galactose, P188, and PLGA to achieve active liver-targeting properties. RBG-loaded Gal-SP188–PLGA nanoparticles (RGPPNs) and coumarin-6-loaded Gal-SP188–PLGA nanoparticles (CGPPNs) were prepared. Thein vitrocellular uptake, cytotoxicity, and apoptosis of nanoparticles in HepG2 cells were analyzed. Thein vivotherapeutic effects of nanoparticles were assessed in a hepatocarcinogenic mouse model. The results showed that Gal-SP188–PLGA was successfully synthesized. The cellular uptake assay demonstrated that CGPPNs had superior active liver-targeting properties. The ratio of apoptotic cells was increased in the RGPPN group. In comparison to the other groups, RGPPNs showed superiorin vivotherapeutic effects and anticancer efficacy. Thus, the active liver-targeting RGPPNs, which can enhance the pharmacological effects and decrease the toxicity of RBG, are expected to become a promising and effective treatment for liver cancer. [ABSTRACT FROM AUTHOR] more...

A novel type of macromolecular prodrug delivery system is reported in this research. The N-galactosylated-chitosan-5-fluorouracil acetic acid conjugate (GC-FUA) based nanoparticle delivery system was evaluatedin vitroandin vivo. Biocompatibility of GC-FUA-NPs was screened by BSA adsorption test and hemolysis activity examinationin vitro. Cytotoxicity and cellular uptake study in HepG2 and A549 cells demonstrated that compared to free 5-Fu, the GC-FUA-NPs play great function in killing cancer cells for the cell endocytosis mediated by asialoglycoprotein receptor (ASGPR), which overexpresses on the cell surface. Pharmacokinetics study further illustrated that the drug-loaded nanoparticles has a much longer half-time than free 5-Fu in blood circulation in Sprague–Dawley (SD) rats. Tissue distribution was investigated in Kunming mice, and the result showed that the GC-FUA-NPs have a long circulation effect. The obtained data suggested that GC-FUA-NP is a very promising drug delivery system for efficient treatment of hepatocellular carcinoma. [ABSTRACT FROM AUTHOR] more...

We report asialoglycoprotein receptor (ASGPR)-targeted doxorubicin hydrochloride (Dox) nanoparticles (NPs) for hepatocellular carcinoma (HCC). Polyethylene sebacate (PES)-Gantrez® AN 119 Dox NPs of average size 220 nm with PDI < 0.62 and ∼20% Dox loading were prepared by modified nanoprecipitation. ASGPR ligands, pullulan (Pul), arabinogalactan (AGn), and the combination (Pul-AGn), were anchored by adsorption. Ligand anchoring enabled high liver uptake with a remarkable hepatocyte:nonparenchymal cell ratio of 85:15. Furthermore, Pul-AGn NPs exhibited an additive effect implying incredibly high hepatocyte accumulation. Galactose-mediated competitive inhibition confirmed ASGPR-mediated uptake of ligand-anchored NPs in HepG2 cell lines. Subacute toxicity in rats confirmed the safety of the NP groups. However, histopathological evaluation suggested mild renal toxicity of AGn. Pul NPs revealed sustained reduction in tumor volume in PLC/PRF/5 liver tumor-bearing Nod/Scid mice up to 46 days. Extensive tumor necrosis, reduced collagen content, reduction in the HCC biomarker serum α-fetoprotein (p < 0.05), a mitotic index of 1.135 (day 46), and tumor treated/tumor control (T/C) values of <0.42 signified superior efficacy of Pul NPs. Furthermore, weight gain in the NP groups, and no histopathological alterations indicated that they were well tolerated by the mice. The high efficacy coupled with greater safety portrayed Pul Dox NPs as a promising nanocarrier for improved therapy of HCC. [ABSTRACT FROM AUTHOR] more...

Circulating tumor cells (CTCs), as potential cancer biomarkers, play a crucial role in cancer diagnosis, prognosis and treatment. However, there are still significant challenges due to their rarity. In this paper, a label-free and ultrasensitive electrochemical impedance cytosensor was developed by a galactosylated gold-nanoisland (Gal-GNI) biointerface for the capture and detection of rare cancer cells (HepG-2) in whole blood samples. By mimic the natural multivalent biorecognition systems, the biointerface was designed to capture the target cancer cells by the specific binding between galactosyl ligands of Gal-GNI and over-expressed asialoglycoprotein receptors (ASGP-R) on the surface of HepG-2. From the results of electrochemical impedance spectroscopy (EIS) and fluorescence spectroscopy, the nanostructured Gal-GNI provides an improved electron-transfer rate, specific detection for HepG-2, and good biocompatibility for viable captured cells. The capture of HepG-2 to the EIS cytosensor increased the electron-transfer resistance, with a good correlation with the logarithm of the concentration from 1.0 × 10 2 to 1.0 × 10 5 cells mL − 1 , with a low detection limit of 30 cells mL − 1 . This cytosensing strategy has good reproducibility with the RSD of 1.9% in blood samples, indicating that it can be used as a potential noninvasive assay tool for the early diagnosis and therapy of tumor. [ABSTRACT FROM AUTHOR] more...

The asialoglycoprotein receptor (ASGR), which is expressed mainly in hepatocytes, is downregulated in poorly differentiated hepatocellular carcinoma (HCC). Here we investigated the role of ASGR1 in HCC metastasis as well as the possible underlying molecular mechanisms. We found that ASGR1 was downregulated in HCC tissue compared with adjacent non-tumorous liver tissue and that lower ASGR1 expression was associated with higher TNM stage and poorer prognosis in HCC patients. ASGR1 overexpression inhibited hepatoma cell migration and invasion in vitro and in vivo, while ASGR1 knockdown had the opposite effects. Furthermore, ASGR1 interacted directly with human longevity assurance homolog 2 of yeast LAG1 (LASS2). Knockdown of LASS2 attenuated the inhibitory effects of ASGR1 on hepatoma cell migration and invasion in vitro. ASGR1 decreased V-ATPase activity in hepatoma cells, and this was reversed by LASS2 knockdown. Finally, HCC patients with low LASS2 levels had poor prognosis, while those with high ASGR1 and LASS2 levels had better prognosis. Thus, ASGR1 may act as a potential metastasis suppressor in HCC, and the combination of ASGR1 and LASS2 may help predict the prognosis of HCC patients. [ABSTRACT FROM AUTHOR] more...

We have developed active targeting liposomes to deliver anticancer agents to ASGPR which will contribute to effective treatment of hepatocellular carcinoma. Active targeting is achieved through polymeric ligands on the liposome surface. The liposomes were prepared using reverse phase evaporation method and doxorubicin hydrocholoride, a model drug, was loaded using the ammonium sulphate gradient method. Liposomes loaded with DOX were found to have a particle size of 200 nm with more than 90% entrapment efficiency. Systems were observed to release the drug in a sustained manner in acidic pH in vitro. Liposomes containing targeting ligands possessed greater and selective toxicity to ASGPR positive HepG2 cell lines due to specific ligand receptor interaction. Bio-distribution studies revealed that liposomes were concentrated in the liver even after 3 h of administration, thus providing conclusive evidence of targeting potential for formulated nanosystems. Tumor regression studies indicated greater tumor suppression with targeted liposomes thereby establishing superiority of the liposomal system. In this work, we used a novel methodology to guide the determination of the optimal composition of the targeting liposomes: molecular dynamics (MD) simulation that aided our understanding of the behaviour of the ligand within the bilayer. This can be seen as a demonstration of the utility of this methodology as a rational design tool for active targeting liposome formulation. [ABSTRACT FROM AUTHOR] more...

Nanoparticles based on the newly synthesized copolymers of linear PLGA blocked with two TPGS ends and galactosylated TPGS were successfully constructed as carriers of oridonin for liver-targeting. The novel copolymers were characterized by 1 H-NMR and TGA. The drug-loaded nanoparticles were prepared by a nanoprecipitation technique and characterized in terms of physicochemical properties, such as particle size, zeta potential, morphology, encapsulation efficiency, in vitro drug release behavior and physical state of the entrapped drug. The ORI-Gal-PT NPs were found to have the highest antitumor efficacy in comparison with the oridonin solution and non-galactosylated nanoparticles and induced a higher apoptotic rate of tumor cells. The targeting nanoparticles could enhance the therapeutic effect of oridonin by increasing uptake of the nanoparticles through asialoglycoprotein receptor-mediated endocytosis. The ORI-Gal-PT NPs system could be a highly promising drug delivery system to be used in liver cancer therapy. [ABSTRACT FROM AUTHOR] more...

The anticancer nucleoside 5-fluoro 2′-deoxyuridine-5′-phosphate (5-FdU-P) was attached via an amide chain linker to a triantennary GalNAc cluster as a means to deliver the drug to hepatic cells that recognize the amino sugar units. [ABSTRACT FROM AUTHOR] more...

The asialoglycoprotein receptor (ASGPR) is a potential target in the search for hepatic cancer drugs. However, application of ASGPR targeting in the clinic is limited by inefficient synthetic methods for the ligand. In this study, we designed and synthesized a novel galactosylated lipid with a mono-galactoside moiety using a lipase. Then we investigated the optimal reaction conditions and analyzed the targeting ability of liposomes modified with the galactosylated lipid. In an organic phase system, different lipases were used as catalysts to synthesize (5-cholesten-3b-yl) [(4-O-β-D-galactopyranosyl)D-glucitol-6] sebacate (CHS-SE-LA). Variables in enzymatic esterification, such as the type of enzyme and solvent, were explored by single-factor experiments. Optimal reaction conditions were determined through response surface methodology. The (CHS-SE-LA)-incorporated galactosylated liposome containing fluorescent dye was then prepared by thin-film hydration and a HepG2 cell transfection test used to confirm the targeting efficiency of galactosylated liposomes to hepatic cancer cells. The structure of CHS-SE-LA was identified by electrospray ionization or ESI and nuclear magnetic resonance or NMR. Under optimal conditions, the predicted yield of CHS-SE-LA was 94.3%, and the actual experimental value was 95.6 ± 1.35%, n = 3. For HepG2 cells, the cellular fluorescence intensities of liposomes modified with CHS-SE-LA (galactosylated liposomes [GAL-FL]) were as much as 2.6-fold (P < 0.01) the control liposomes (FL). Moreover, the presence of excess galactose significantly inhibited the uptake of GAL-FL suggesting ASGPR mediated uptake. The novel galactosylated ligand was synthesized enzymatically with high efficiency under mild conditions. Liposomes containing CHS-SE-LA have great potential as drug delivery carriers for hepatocyte-selective targeting. [ABSTRACT FROM AUTHOR] more...

Mono-antennary galacto derivatives of cholesterol are being actively developed to direct lipoplexes to the asialoglycoprotein receptor (ASGP-R) on hepatocytes. Here we report on a novel ASGP-R ligand cholest-5-en-3-yl [1-(β- d-galactopyranosyl)-1 H-1,2,3-triazol-4-yl]methylcarbamate ( 4), assembled by a copper(I)-catalyzed azide-alkyne cycloaddition (click chemistry), and compare it with cholest-5-en-3-yl-β- d-galactopyranoside ( 2) and cholest-5-en-3-yl [1-(β- d-galactopyranosyl-1′-oxy)phen-4-yl]carbamate ( 3), in liposome formulations with or without 5 mol% distearoylphosphatidylethanolamine poly(ethylene glycol)2000, intended for DNA delivery to ASGP-R-positive hepatocyte-derived HepG2 cells and the ASGP-R-negative embryo kidney cell line HEK293. Transfection levels attained with lipoplex 4 were 100 and 300 % greater than those for lipoplexes 2 and 3 respectively in HepG2 cells, while competition assays reduced transfection levels by up to 98 %. Transfection activities achieved in HEK293 cells were up to three orders of magnitude lower. Therefore, 4 is representative of a new class of promising hepatotropic ligands for gene delivery. [ABSTRACT FROM AUTHOR] more...

Gene therapy is considered a promising approach for the treatment of hepatocellular carcinoma (HCC). In this regard, the main goal of this work was to develop a specific and efficient gene delivery nanosystem to HCC based on 1-palmitoyl-2-oleoyl- sn -glycero-3-ethylphosphocholine:cholesterol cationic liposomes and asialofetuin (ASF), a specific ligand to the asialoglycoprotein receptor (ASGP-R) that is overexpressed in HCC. Our results show that association of ASF to lipoplexes promotes a substantial increase in their biological activity in HCC cells, not only in vitro, but also in an animal model. The transfection activity obtained with this novel nanosystem (ASF-lipoplexes) was much higher than that observed with a highly efficient commercial formulation. On the other hand, the presence of high concentrations of galactose substantially reduced the cell uptake and biological activity of the ASF-lipoplexes. These results, together with those obtained in the presence of inhibitors of endocytosis, show that the potentiation induced by the association of ASF to lipoplexes is due to its specific interaction with the ASGP-R. The physicochemical properties of the generated nanosystem also reinforce this observation. Overall, our results demonstrate for the first time that the novel ASF-lipoplexes present a noticeable ability to specifically and efficiently deliver genetic material into HCC cells. [ABSTRACT FROM AUTHOR] more...

Background: A specific targeting modality for hepatocellular carcinoma (HCC) could ideally encompass a liver cell specific delivery system of a transcriptional unit that is active only in neoplastic cells. Sendai virosomes, derived from Sendai viral envelopes, home to hepatocytes based on the liver specific expression of asialoglycoprotein receptors (ASGPRs) which are recognized by the Sendai virosomal fusion (F) proteins. As reported earlier by us and other groups, transcriptional gene silencing (TGS) does not require continuous presence of the effector siRNA/shRNA molecule and is heritable, involving epigenetic modifications, leading to long term transcriptional repression. This could be advantageous over conventional gene therapy approaches, since continuous c-Myc inactivation is required to suppress hepatocarcinoma cells. Methods: Exploiting such virosomal delivery, the alpha-fetoprotein (AFP) promoter, in combination with various tumour specific enhancers, was used to drive the expression of shRNA directed against ME1a1 binding site of the proto-oncogene c-Myc P2 promoter, in order to induce TGS in neoplastic liver cells. Results: The dual specificity achieved by the Sendai virosomal delivery system and the promoter/enhancer guided expression ensured that the shRNA inducing TGS was active only in liver cells that had undergone malignant transformation. Our results indicate that such a bimodal therapeutic system induced specific activation of apoptosis in hepatocarcinoma cells due to heterochromatization and increased DNA methylation of the CpG islands around the target loci. Conclusions: The Sendai virosomal delivery system, combined with AFP promoter/enhancer expression machinery, could serve as a generalized mechanism for the expression of genes deleterious to transformed hepatocarcinoma cells. In this system, the epigenetic suppression of c-Myc could have an added advantage for inducing cell death in the targeted cells. [ABSTRACT FROM AUTHOR] more...

In some cancer cells, translocation of cytochrome c (Cyt c) from mitochondria to the cytoplasma is inhibited. This inhibition prevents cells from undergoing apoptotic cell death and can lead to uncontrolled cell growth. Increasing cytoplasmic concentration of Cyt c can induce apoptosis in cancer cells as a strategy of cancer therapy. Here we proposed a galactosylated albumin based carrier for intracellular delivery of Cyt c to hepatocarcinoma cells. Galactosylated albumin is recognized by highly expressed asialoglycoprotein receptors (ASGPR) on hepatocarcinoma cells and is further internalized into cells via receptor mediated endocytosis. Cyt c was chemically conjugated to galactosylated albumin with a reducible disulfide linker in order to release Cyt c from the carrier inside cells. We tested cellular uptake and cytotoxicity of Cyt c conjugates in ASGPR positive and negative hepatocarcinoma cells. The results showed galatosylated albumin significantly increased cellular uptake in both cell types resulting in cytotoxicity in a dose dependent manner through the induction of apoptosis. The lack of ASGPR specific uptake might be due to other carbohydrate-recognizing receptors expressed on tumor cells. In general, our work has shown that intracellular delivery of Cyt c to tumor cells can be an alternative therapeutic approach and galactosylated albumin can be a protein drug carrier for intracellular delivery. [ABSTRACT FROM AUTHOR] more...

Background and purpose: The hepatocyte asialoglycoprotein receptor mediates uptake of desiaylated glycoproteins by receptor-mediated endocytosis. This work explores a hepatocyte-specific targeting strategy using asialofetuin (AF) covalently coupled to pegylated liposomes. Methods: AF was conjugated to the distal end of polyethylene glycol-functionalized phospholipids. Chemical modification of AF did not interfere with its receptor interaction. AF-liposomes had a size of less than 130 nm, were judged to be monodisperse and were labelled with fluorescent organic dyes or loaded with quantum dots. Results: In vitro, binding and cellular uptake of fluorescent AF-liposomes by HepG2 hepatocellular carcinoma cells were reduced at low temperature and could be competitively inhibited by an excess of unbound AF. Hepatocyte-specific targeting and internalization of AF-liposomes in vivo was confirmed in the rat and could be competitively inhibited by co-injection of unbound AF. In contrast, non-pegylated liposomes accumulated in cells of the reticuloendothelial system such as hepatic Kupffer cells and spleen after intravenous administration. Conclusion: We conclude that the use of AF-conjugated, pegylated liposomes is a promising strategy to avoid the reticuloendothelial system and specifically target hepatocytes via the asialoglycoprotein receptor in vitro as well as in vivo. [ABSTRACT FROM AUTHOR] more...

Background: Asialoglycoprotein receptor (ASGPR)-ligand-based separation combined with identification with Hep Par 1 or pan-cytokeratin (P-CK) antibody have been demonstrated to detect circulating tumor cells (CTCs) in hepatocellular carcinoma (HCC). The aim of this study was to develop an improved enrichment and identification system that allows the detection of all types of HCC CTCs. Methods: The specificity of the prepared anti-ASGPR monoclonal antibody was characterized. HCC cells were bound by ASGPR antibody and subsequently magnetically isolated by second antibody-coated magnetic beads. Isolated HCC cells were identified by immunofluorescence staining using a combination of anti-P-CK and anti-carbamoyl phosphate synthetase 1 (CPS1) antibodies. Blood samples spiked with HepG2 cells were used to determine recovery and sensitivity. CTCs were detected in blood samples from HCC patients and other patients. Results: ASGPR was exclusively expressed in human hepatoma cell line, normal hepatocytes and HCC cells in tissue specimens detected by the ASGPR antibody staining. More HCC cells could be identified by the antibody cocktail for CPS1 and P-CK compared with a single antibody. The current approach obtained a higher recovery rate of HepG2 cells and more CTC detection from HCC patients than the previous method. Using the current method CTCs were detected in 89% of HCC patients and no CTCs were found in the other test subjects. Conclusions: Our anti-ASGPR antibody could be used for specific and efficient HCC CTC enrichment, and anti-P-CK combined with anti-CPS1 antibodies is superior to identification with one antibody alone in the sensitivity for HCC CTC detection. [ABSTRACT FROM AUTHOR] more...

Highlights: [•] PVA microbubbles are coated with chitosan-galactosylated (GC) to target hepatocarcinoma. [•] GC confer specificity for asialoglycoprotein receptor (ASGPR). [•] GC-microbubbles show bioadhesive properties/selectivity for tumor cells expressing ASGPR. [•] Doxorubicin-loaded particles display higher cytotoxic activity than the free drug. [•] Cytotoxicity of GC-microbubbles /doxorubicin is increased upon exposure to ultrasound. [Copyright &y& Elsevier] more...

Background: Asialoglycoprotein receptors are among the most important over-expressed receptors in hepatocellular carcinoma (HepG2). Decorating drug carrier to specific ligands of these receptors can enhance their absorption to cells and elevate their permeability to hepatoma cells. This can reduce the side effects of the drug in nonspecific tissues and enhance drug efficiency. The aim of the present work was to evaluate the cytotoxicity of doxorubicin-loaded mixed micelles of galactosylated Pluronic F127/L122 on HepG2 cells. Methods: Galactosylated Pluronic F127 was synthesized. Doxorubicin-loaded nano-sized micelles were prepared by direct dissolution method. Based on particle size, zeta potential, polydispersity index, loading efficiency and release efficiency of doxorubicin from nano-sized micelles, optimized micelles were used for cytotoxicity test on HepG2 cells by 3-(4,5-dimethythiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay. Findings: The optimum targeted nano-sized micelles had particle size of 197.9 ± 2.1 nm, zeta potential of 18.6 mv, polydispersity index of 0.308 ± 0.058, loading efficiency of 78.3 ± 0.3%, and drug release efficiency of 32.5 ± 0.5% (until the third hour). Nano-sized micelles loaded with doxorubicin in concentrations of 0.2, 0.8, 3, 6 μM caused significantly higher cytotoxicity compared to non-targeted nano-sized micelles and free doxorubicin. Conclusion: Mixed targeted nano-sized micelles of galactosylated Pluronic F127 and Pluronic L122 loaded with doxorubicin showed more efficiency and specificity to toxicity against HepG2 cells than non-targeted nanoparticles and free drug. [ABSTRACT FROM AUTHOR] more...

Abstract: Early metastases of hepatocellular carcinoma (HCC) may be detected by the isolation of circulating tumor cells (CTCs) in the bloodstream. During the course of therapeutic attempts, monitoring CTC changes in patients with HCC is helpful for the efficacy assessment. Nevertheless, the markers used for the detection, such as α-feto protein, asialoglycoprotein receptor or epithelial cell adhesion molecule, CD133 or CD90, are not specific for HCC CTCs. In spite of these limitations, a timely determination of the existence of CTCs will be beneficial for the monitoring of distant metastases, the evaluation of therapeutic attempts, and the prediction of prognosis. [Copyright &y& Elsevier] more...

Liposomes form a major class of non-viral vectors for short interfering RNA delivery, however tissue and cell-specific targeting are additional requirements in the design of short interfering RNA delivery systems with a therapeutic potential. Selective delivery of liposomes to hepatocytes may be achieved by directing complexes to the asialoglycoprotein receptor, which is expressed on hepatocytes, and which displays high affinity for the β- d-galactopyranosyl moiety. We aimed to show that the d-galactopyranosyl ring in direct β-glycosidic link to cholesterol, when formulated into liposomes with 3β[ N-( N′, N′-dimethylaminopropane) carbamoyl] cholesterol (Chol-T) or its quaternary trimethylammonium analogue (Chol-Q), may promote targeted delivery of cytotoxic short interfering RNA to the human hepatoma cell line HepG2 via the asialoglycoprotein receptor. Liposome-short interfering RNA interactions were characterized by electron microscopy, dye displacement, gel retardation and nuclease assays. Stable short interfering RNA-protective lipoplexes were formed at N/P ratios in the range 5:1-7:1. Targeted lipoplex 4 achieved high transfection efficiencies at 50 n m short interfering RNA (70%) and <10% in a competition assay, whilst untargeted complexes reached low levels at the same concentration (<25%). Transfection efficiencies of all lipoplexes in the asialoglycoprotein receptor-negative cell line HEK293 under the same conditions were low. Lipoplexes containing cholesteryl-β- d-galactopyranoside may therefore form the basis for the development of useful hepatotropic short interfering RNA delivery vectors. [ABSTRACT FROM AUTHOR] more...

Hepatitis B virus (HBV) infection is a worldwide liver disease and nearly 25% of chronic HBV infections terminate in hepatocellular carcinoma (HCC). Currently, there is no effective therapy to inhibit HBV replication and to eliminate hepatoma cells, making it highly desired to develop novel therapies for these two stages of the HBV-caused detrimental disease. Recently, short hairpin RNA (shRNA) has emerged as a potential therapy for virus-infected disease and cancer. Here, we have generated a shRNA, pGenesil-siHBV4, which effectively inhibits HBV replication in the human hepatoma cell line HepG2.2.15. The inhibitory effects of pGenesil-siHBV4 are manifested by the decrease of both the HBV mRNA level and the protein levels of the secreted HBV surface antigen (HBsAg) and HBV e antigen (HBeAg), and by the reduction of secreted HBV DNA. Using mouse hydrodynamic tail vein injection, we demonstrate that pGenesil-siHBV4 is effective in inhibiting HBV replication in vivo. Because survivin plays a key role in cancer cell escape from apoptosis, we further generated pGenesil-siSurvivin, a survivin-silencing shRNA, and showed its effect of triggering apoptosis of HBV-containing hepatoma cells. To develop targeted shRNA therapy, we have identified that as a specific binder of the asialoglycoprotein receptor (ASGPR), jetPEI-Hepatocyte delivers pGenesil-siHBV4 and pGenesil-siSurvivin specifically to hepatocytes, not other types of cells. Finally, co-transfection of pGenesil-siHBV4 and pGenesil-siSurvivin exerts synergistic effects in inducing hepatoma cell apoptosis, a novel approach to eliminate hepatoma by downregulating survivin via multiple mechanisms. The application of these novel shRNAs with the jetPEI-Hepatocyte targeting strategy demonstrates the proof-of-principle for a promising approach to inhibit HBV replication and eliminate hepatoma cells with high specificity. [ABSTRACT FROM AUTHOR] more...

Targeted delivery of drugs and proteins to liver can be achieved via asialoglycoprotein receptor, which can recognize and combine the galactose- and N-acetygalatosamineterminated glycoproteins. Glycosyl is usually conjugated with drugs directly to fabricate prodrugs or with nanoparticles encapsulated drugs via forming covalent bonds, while the covalent bonds may lead to some shortages for drug release. Therefore, we have a hypothesis that we can prepare nanoparticles for efficient targeting by glycosylation using galactosylated poly (L-glutamic acid) (Gal-PLGA) as a carrier to entrap the model drugs in nanoparticles core physically rather than forming covalent drug conjugation. The means of incorporation of drug in nanoparticles may improve drug release to maintain its activity, raise its therapeutic index and diminish the adverse effect. Based on previous researches, it is achievable to obtain nanoparticles that we hypothesize to prepare. Due to their nanometer-size and galactosyl, the nanoparticles may be a potential delivery system for passive and active targeting to liver parenchymal cells for therapy of hepatitis and liver cancer. [ABSTRACT FROM AUTHOR] more...