In vitro targeting effect of lactoferrin modified PEGylated liposomes for hepatoma cells / 药学学报

A lactoferrin-containing PEGylated liposome system (Lf-PLS) was developed and tested in vitro as a hepatoma-targeting drug delivery system. PEGylated liposomes (PLS) were successfully prepared using the thin film hydration method with peglipid post insertion. Lf was covalently conjugated onto the carboxyl terminal of DSPE-PEG2000-COOH on liposomes. Coumarin-6 was used to trace Lf-PLS with fluorescence. The cellular uptake of this system was carried out in asialoglycoprotein receptor (ASGPR) positive HepG2 cells via confocal microscopy and flow cytometry. The Lf-PLS liposome was observed as spherical or oval vesicles with the particle size around 130 nm, zeta potential about -30 mV and encapsulation efficiency more than 80%. The confocal microscopy images and flow cytometry data demonstrated that Lf-PLS resulted in significantly higher cell association by ASGPR positive HepG2 cells compared to PLS. The association between Lf-PLS and cells were dependent on the concentration, time and temperature, which was inhibited by pre-incubation with excessive free Lf. The results suggest that Lf-PLS has a good targeting effect on HepG2 cells in vitro. The targeting mechanism may be related to the specific binding of Lf and ASGPR on HepG2 cells, which guides Lf-PLS to the cell surface to induce an active endocytosis process. All these results demonstrated that Lf-PLS might be a potential drug delivery system in targeting hepatocellular carcinoma, which deserves more research on its targeting ability, antitumor efficiency, and metabolism in vivo for treatment of hepatomacellular carcinoma.

Galactosylated albumin nanoparticles of simvastatin

The present study was an attempt to develop galactosylated albumin nanoparticles of Simvastatin for treatment of hypercholesterolemia. By developing the galactosylated nanoparticulated delivery, the required action of the drug at the target site at the liver can be provided. The advantage of targeting helps to reduce the systemic side effects that may occur due to the distribution of the drug to the other organs and thus helps in maintaining the required concentration of drug at the desired site. The galacotsylated albumin nanoparticles were prepared for the selective delivery of a Simvastatin to the 3-hydroxy-3-methylglutaryl coenzyme A reductase [HMG-CoA reductase] the rate-limiting enzyme in the pathway of cholesterol biosynthesis that is particularly presents on hepatocytes. The asialoglycoprotein receptor [ASGP-R] which is particularly presents on mammalian hepatocytes can be utilize for active targeting by using its natural and synthetic ligands. By utilizing this receptors can provides a unique means for the development of liver-specific carriers, such as liposomes, recombinant lipoproteins, and polymers for drug or gene delivery to the liver, especially to hepatocytes. These receptors recognize the ligands with terminal galactose or N-acetylgalactosamine residues, and endocytose the ligands for an intracellular degradation process. The albumin nanoparticles [NPs] were prepared by using desolvation method and efficiently conjugated with galactose. Various parameters such as particle size, zeta potential, percentage entrapment efficiency and drug loading efficiency, percentage yield, in-vitro drug release were determined. The size of nanoparticles [both plain and coated NPs] was 200 and 250 nm. The zeta potential of plain nanoparticles was -3.61 and that of galactose-coated nanoparticles was 64.1. The maximum drug content was in between 79.98% to 79.8 % respectively in plain, and galactose coated nanoparticles while the maximum entrapment efficiency was 70.10% and 71.03% in plain and coated nanoparticles. It was found that coating of nanoparticles increases the size of nanoparticles

Enzyme-catalyzed synthesis of ASGPR ligand-targeted modifier in non-aqueous medium / 药学学报

The asialoglycoprotein receptor (ASGPR) was used to mediate drug carrier for hepatic targeted drug delivery, this article showed the enzyme-catalyzed esterification of galactose and vinyl stearate and a kind of ASGPR ligand-targeted which was used to insert the surface of liposome has been synthesized. The structure of product has been confirmed by TLC, ESI-MS and 1H NMR. The factors of types and quantity of enzyme, organic solvents, molar ratio of substrate, temperature and time of reaction have been studied. Results showed when using acetone as reaction medium, the quantity of Novozym 435 immobilized lipase was 30 mg mL(-1), molar ratio of galactose to vinyl stearate was 1:5, and reacted at 60 degrees C for 12 h, the transformation of vinyl stearate reached more than 70%. This study provides a novel and efficient route to the synthesis of ligand-targeted modifier.

Synthesis of Gal-BSA-SPIO and magnetic resonance imaging of ASG receptors in rabbits bearing liver VX2 tumor and human liver / 南方医科大学学报

<p><b>OBJECTIVE</b>To synthesize Gal-BSA-SPIO as the magnetic resonance imaging (MRI) contrast agent targeting asialoglycoprotein (ASG) receptors in the liver and observe its role in MRI detection of hepatocellular carcinomas (HCCs).</p><p><b>METHODS</b>Gal-BSA was synthesized by means of reductive amination and mixed with SPIO in ice bath to prepare Gal-BSA-SPIO complex. Twenty rabbits bearing VX2 liver tumor underwent MRI enhanced by SPIO (n=10) and Gal-BSA-SPIO (n=10), and the T2 values of the liver and tumor before and after the contrast imaging were measured. Fresh human normal hepatic tissues (n=3), cirrhotic tissues (n=4) and HCC tissues (n=6) were obtained and incubated with Gal-BSA-SPIO followed by Perl's Prussian blue staining to observe the distribution of ASG receptors.</p><p><b>RESULTS</b>The size of the Gal-BSA-SPIO particles was 34.4 nm. The 20 rabbits bearing VX2 tumor, with tumor size ranging from 3 mm to 12 mm, showed isointense signal in the liver and hypointense signal in the tumor on T1WI, and isointense signal in the liver and slightly hyperintense signal in the tumor on GRE T2*WI. The signal intensity of the liver decreased slightly or moderately after administration of SPIO in the rabbits, and administration Gal-BSA-SPIO resulted in obvious reduction in the signal intensity of the liver. The signal intensities of the tumors did not exhibit obvious changes after the administration of SPIO or Gal-BSA-SPIO. Histological examination revealed numerous blue iron deposits in the Kupffer cells in SPIO group and in the hepatocytes in Gal-BSA-SPIO group, but not in the tumors in either of the groups. The human liver specimens incubated with Gal-BSA-SPIO contained numerous blue iron deposits in the hepatocyte cytoplasm and cell membrane in normal liver tissue, but the deposits were reduced in the cirrhotic tissue and almost absent in the HCC tissue.</p><p><b>CONCLUSION</b>Gal-BSA-SPIO can specifically bind to ASG receptors on hepatocyte membrane to improve the tumor-liver contrast-to-noise ratio.</p>

Biodistribution of (99m) Tc-Lactosylated Serum Albumin in Mice with Diethylnitrosamine or Thiacetamide Induced Liver Injury / 대한핵의학회잡지

PURPOSE: Tc-99m labeled diethylenetriaminepentaacetic acid (DTPA) -coupled galactosylated human serum albumin (GSA) is a currently used imaging agent for asialoglycoprotein receptor (ASGPR) of the liver, but, it has several shortcomings. Recently a new ASGPR imaging agent, (99m) Tc-lactosylated human serum albumin (LSA), with simple labeling procedure, high labeling efficiency, high stability was developed. In order to assess the feasibility of the (99m) Tc-LSA as a ASGPR imaging radiopharmaceuticals, we performed biodistribution study of the tracer in liver injured mice model and the results were compared with histolgic data. MATERIALS AND METHODS: To induce hepatic damage in ICR mice, diethylnitrosamine (DEN) (60 mg/kg/week X 5 time, low dose or 180 mg/kg/week X 2 times, high dose) and thioacetamide (TAA) (50 mg/kg X 1 time) were administrated intraperitoneally. Degree of liver damage was evaluated by tissue hematoxilin-eosin stain, and expression of asialoglycoprotein receptor (ASGPR) was assessed by immunohistochemistry using ASGPR antibody. (99m) Tc-LSA was intravenously administrated via tail vein in DEN or TAA treated mice, and biodistribution study of the tracer was also performed. RESULTS: DEN treated mice showed ballooning of hepatocyte and inflammatory cell infiltration in low dose group and severe hapatocyte necrosis in high dose group, and low dose group showed higher ASGPR staining than control mice in immunohistochemical staining. TAA treated mice showed severe hepatic necrosis. (99m) Tc-LSA Biodistribution study showed that mice with hepatic necrosis induced by high dose DEN or TAA revealed higher blood activity and lower liver activity than control mice, due to slow clearance of the tracer by the liver. The degree of liver uptake was inversely correlated with the degree of histologic liver damage. But low dose DEN treated mice with mild hepatic injury showed normal blood clearance and hepatic activity, partly due to overexpression of ASGPR in mice with mild degree hepatic injury. CONCLUSION: Liver uptake of (99m) Tc-LSA was inversely correlated with degree of histologic hepatic injury in DEN and TAA treated mice. These results support that (99m) Tc-LSA can be used to evaluate the liver status in liver disease patients.

Evaluation of Liver Function Using 99mTc-Lactosylated Serum Albumin Liver Scintigraphy in Rat with Acute Hepatic Injury Induced by Dimethylnitrosamine / 대한핵의학회잡지

OBJECTS: 99mTc-lactosylated human serum albumin (LSA) is a newly synthesized radiopharmaceutical that binds to asialoglycoprotein receptors, which are specifically presented on the hepatocyte membrane. Hepatic uptake and blood clearance of LSA were evaluated in rat with acute hepatic injury induced by dimethylnitrosamine (DMN) and results were compared with corresponding findings of liver enzyme profile and these of histologic changes. MATERIALS AND METHODS: DMN (27 mg/kg) was injected intraperitoneally in Sprague-Dawley rat to induce acute hepatic injury. At 3 (DMN-3), 8 (DMN-8), and 21 (DMN-21) days after injection of DMN, LSA injected intravenously, and dynamic images of the liver and heart were recorded for 30 minutes. Time-activity curves of the heart and liver were generated from regions of interest drawn over liver and heart area. Degree of hepatic uptake and blood clearance of LSA were evaluated with visual interpretation and semiquantitative analysis using parameters (receptor index : LHL3 and index of blood clearance : HH3), analysis of time-activity curve was also performed with curve fitting using Prism program. RESULTS: Visual assessment of LSA images revealed decreased hepatic uptake in DMN treated rat, compared to control group. In semiquantitative analysis, LHL3 was significantly lower in DMN treated rat group than control rat group (DMN-3: 0.842, DMN-8: 0.898, DMN-21: 0.91, Control: 0.96, p< 0.05), whereas HH3 was significantly higher than control rat group (DMN-3: 0.731, DMN-8: 0.654, DMN-21: 0.604, Control: 0.473, p< 0.05). AST and ALT were significantly higher in DMN-3 group than those of control group. Centrilobular necrosis and infiltration of inflammatory cells were most prominent in DMN-3 group, and were decreased over time. CONCLUSION: The degree of hepatic uptake of LSA was inversely correlated with liver transaminase and degree of histologic liver injury in rat with acute hepatic injury.

Study on the hepatocytic cell targetability of liposomes / 药学学报

<p><b>AIM</b>To target for hepatocytic cell, liposomes was modified by special ligand.</p><p><b>METHODS</b>Sterically stabilized liposomes (SSL) was conjugated with asialofeticin (AF), the ligand of asialoglycoprotein receptor (ASGP-R) of hepatocyte. ASGP-R-BLM is the ASGP-R reconstructed on bilayer lipid membrane (BLM). The recognition reaction between AF-SSL and ASGP-R-BLM can be monitored by the varieties of membrane electrical parameters. The targetability of AF-SSL mediated to hepatocyte was detected by radioisotopic labeled in vitro and in vivo. The therapeutic effect of antihepatocarcinoma was observed also.</p><p><b>RESULTS</b>The lifetime of ASGP-R-BLM decreased with the added amount of AF-SSL. It was demonstrated that there was recognition reaction between AF-SSL and ASGP-R-BLM. The combination of AF-SSL with hepatocyte was significantly higher than that of SSL without AF-modified in vitro and in vivo. The survival time of rat for AF-SSL carriered ADM (adriamycin) group was much longer and the toxicities on heart, kidney and lung were lower than those SSL carried ADM group.</p><p><b>CONCLUSION</b>It is possible to actively target the cell with specific receptor by ligand modified liposomes. The result prvide scientific basis of hepatocyte targeted liposomes.</p>

99mTc-DTPA Galactosyl Human Serum Albumin Scintigraphy in Mushiroom Poisoning Patient: Comparison with Liver Ultrasonography / 대한핵의학회잡지

99mTc-galactosyl human serum albumin (Tc-GSA) is a radiopharmaceutical that binds to asialoglycoprotein receptors, which are specifically present in the hepatocyte membrane. Because these receptors are decreased in hepatic parenchymal damage, the degree of Tc-GSA accumulation in the liver correlates with findings of liver function test. Hepatic images were performed with Tc-GSA in patients with acute hepatic dysfunction by Amantia Subjunquillea poisoning, and compared with these of liver ultrasonography (USG). Tc-GSA (185 MBq, 3 mg of GSA) was injected intravenously, and dynamic images were recorded for 30 minutes. Time-activity curves for the heart and liver were generated from regions of interest for the whole liver and precordium. Degree of hepatic uptake and clearance rate of Tc-GSA were generated by visual interpretation and semiquantitative analysis parameters (receptor index: LHL15 and index of blood clearance: HH15). Visual assessment of GSA scintigraphy revealed mildly decreased liver uptake in all of subjects. The mean LHL15 and HH15 were 0.886 and 0.621, graded as mild dysfunction in 2, and mild to moderate dysfunction in 1 subject. In contrast, liver USG showed no remarkable changes of hepatic parenchyme. Tc-GSA scintigraphy was considered as a useful imaging modality in the assessment of the hepatic dysfunction.

Quantitative Evaluation of Liver Function with Hepatic Receptor Scintigraphy using Tc-99m Galactosylated Serum Albumin ( GSA ) / 대한핵의학회잡지

The reduction in the amount of asialoglycoprotein (ASGP) receptor, which resides exclusively on the plasma membrane of functioning mammalian hepatocytes, as a consequence of hepato-cellular damage has been demonstrated in various pathologic conditions of the liver. Galac tosylated human serum albumin (GSA) is a newly developed receptor-binding agent, specific for the ASGP receptor. Tc-99m GSA binds quantitatively to liver ASGP receptors and the rate of accumulation in the liver is dependent on hepatic function represented as the amount of receptor, as well as the amount of ligand injected, its affinity to the receptor and the hepatic blood flow. The findings of Tc-99m GSA scintigraphy were reported to reflect the hepatic function of the patients with large hepatic tumors, obstructive jauniice, acute and chronic liver disease. Tc-99m GSA scintigraphy is an easy and reliable test and has the clinical potentials to evaluate the liver function in the patients with hepatic disorders.

Hepatic Asialoglycoprotein Receptor Scintigraphy Using 99mTc - Galactosylated Serum Albumin ( GSA ): Biodistribution Study in Mice with Thioacetamide - induced Hepatic Injury / 대한간학회지

BACKGROUND/AIMS: Tc-DTPA-galactosylated serum albumin (GSA) is a new liver imaging agent that specifically binds to asialoglycoprotein receptor, which resides exclusively on the plasma membrane of mammalian hepatocytes. To evaluate the usefulness of hepatic scintigraphy with Tc-GSA in the assessment of hepatic function, we have investigated serial changes in organ uptakes of Tc-GSA in mice with thioacetamide-induced hepatic injury and compared to changes in histology or levels of hepatic enzymes. MATERIALS AND METHODS: Acute hepatic injuries were induced by intraperitoneal injection of thioacetamide in ICR (Institute of Cancer Research) mice. Hepatic injuries were serially assessed by either light microscopic examination of liver slices or measurement of hepatic enzymes. The biodistribution of Tc-GSA was measured in liver and each excised organs using gamma counter. RESULTS: Hepatic injuries in light microscopic examination were not evident at 12 hours after injection of thioacetamide but resulted in maximal centrilobular necrosis and inflammation at 24 hours. These histologic changes were progressively improved upto 72 hours. Studied hepatic enzymes were elevated at 6 hours and reached to maximal level at 24 hours after administration of thioacetamide and declined progressively after then. Hepatic uptake of Tc-GSA was lowest at 6 hours after administration of thioacetamide and serially recovered until 72 hours. CONCLUSION: The degree in the decrease of hepatic uptake of Tc-GSA was precedent to either histologic changes or elevation of hepatic enzymes and correlated with the degree of hepatic daneges in acute hepatic injury. These findings suggest that hepatic scintigraphy using Tc-GSA can be used to detect early changes and serial assessment of hepatic function in acute hepatic injury.

Hepatic Asialoglycoprotein Receptor Scintigraphy Using 99mTc - Galactosylated Serum Albumin ( GSA ): Biodistribution Study in Mice with Thioacetamide - induced Hepatic Injury / 대한간학회지

BACKGROUND/AIMS: Tc-DTPA-galactosylated serum albumin (GSA) is a new liver imaging agent that specifically binds to asialoglycoprotein receptor, which resides exclusively on the plasma membrane of mammalian hepatocytes. To evaluate the usefulness of hepatic scintigraphy with Tc-GSA in the assessment of hepatic function, we have investigated serial changes in organ uptakes of Tc-GSA in mice with thioacetamide-induced hepatic injury and compared to changes in histology or levels of hepatic enzymes. MATERIALS AND METHODS: Acute hepatic injuries were induced by intraperitoneal injection of thioacetamide in ICR (Institute of Cancer Research) mice. Hepatic injuries were serially assessed by either light microscopic examination of liver slices or measurement of hepatic enzymes. The biodistribution of Tc-GSA was measured in liver and each excised organs using gamma counter. RESULTS: Hepatic injuries in light microscopic examination were not evident at 12 hours after injection of thioacetamide but resulted in maximal centrilobular necrosis and inflammation at 24 hours. These histologic changes were progressively improved upto 72 hours. Studied hepatic enzymes were elevated at 6 hours and reached to maximal level at 24 hours after administration of thioacetamide and declined progressively after then. Hepatic uptake of Tc-GSA was lowest at 6 hours after administration of thioacetamide and serially recovered until 72 hours. CONCLUSION: The degree in the decrease of hepatic uptake of Tc-GSA was precedent to either histologic changes or elevation of hepatic enzymes and correlated with the degree of hepatic daneges in acute hepatic injury. These findings suggest that hepatic scintigraphy using Tc-GSA can be used to detect early changes and serial assessment of hepatic function in acute hepatic injury.