AIM: To investigate the uptake difference between bovine serum albumin nanoparticle (BSA-NP) and bovine serum albumin nanoparticles with their surface modified byglycyrrhizin (BSA-NP-GL) and to develop a novel hepatoc...AIM: To investigate the uptake difference between bovine serum albumin nanoparticle (BSA-NP) and bovine serum albumin nanoparticles with their surface modified byglycyrrhizin (BSA-NP-GL) and to develop a novel hepatocyte targeting BSA-NP-GL based on active targeting technology mediated by specific binding site of GL on rat cellular membrane. METHODS: Calcein loaded bovine serum albumin nanoparticles (Cal-BSA-NP) were prepared by desolvation process. Glycyrrhizin was conjugated to the surface reactive amino groups (SRAG) of Cal-BSA-NP by sodium periodate oxidization, which resulted in calcein-loaded bovine serum albumin nanoparticles with their surface modified by glycyrrhizin (Cal-BSA-NP-GL). The morphology of the two types of prepared nanoparticles (NP) was observed by transmission electron microscopy. The diameter of NP was measured with a laser particle size analyzer. The interaction between Cal-BSA-NP-GL and primary cultured hepatocytes was studied through cellular uptake experiments. The uptake amount of Cal-BSA-NPGL and Cal-BSA-NP by rat hepatocytes was determinedby fluorospectrophotometry. Uptake characteristics were investigated through experiments of competitive inhibition of specific binding site of GL. RESULTS: Both Cal-BSA-NP-GL and Cal-BSA-NP had regular spherical surfaces. The average diameter of CalBSA-NP-GL and Cal-BSA-NP was 77 and 79 nm respectively. The uptake amount of the two NP by hepatocytes reached its maximum at 2 h after incubation. The uptake amount of Cal-BSA-NP-GL by rat hepatocytes was 4.43-fold higher than that of Cal-BSA-NP. There was a significant difference in the uptake of Cal-BSA-NP-GL and Cal-BSA-NP by hepatocytes (P<0.01). The uptake of Cal-BSA-NP-GL was inhibited when GL was added previously to isolated rat hepatocytes, and the uptake of Cal-BSA-NP was not affected by GL.CONCLUSION: A binding site of GL is present on the surface of rat hepatocytes, BSA-NP-GL may be internalized via this site by hepatocytes and can be used as a drug carrier for active targeting of delivery drugs to hepatocytes.展开更多
AIM:To develop a novel non-viral gene delivery system,which has a small particle size and a high transfection efficiency to hepatocyte and hepatoma cells.METHODS:Lipid-polycation-DNA lipopolyplex (LPD) was prepared by...AIM:To develop a novel non-viral gene delivery system,which has a small particle size and a high transfection efficiency to hepatocyte and hepatoma cells.METHODS:Lipid-polycation-DNA lipopolyplex (LPD) was prepared by mixing plasmid DNA and polylysine. The resulted polyplex was incubated for 10min at room temperature,following the addition of preformed cationic liposomes. The morphology of LPD was observed by transmission electron microscopy.The diameter and surface charge of LPD were measured by photon correlation spectroscopy (PCS). The nuclease protection ability of LPD was evaluated by agarose gel electrophoresis. Estimation of the transfection efficiency was performed by galactosidase assay in Chang cells and SMMC-7721 cells.RESULTS:LPD had a regular spherical surface. The average diameter and the zeta potential of LPD were 132.1nm and 26.8mV respectively. LPD could protect plasmid DNA from nuclease degradation after 2 hours incubation at 37℃ while the naked DNA degraded rapidly. The average transfection efficiencies were 86.2±8.9% and 72.4±6.5% in Chang cells and SMMC-7721 cells respectively.CONCLUSION: LPD has a rather small particle size and a high transfection activity. LPD may be a good non-viral vector for application in some gene delivery.展开更多
AIM:To prepare thymidine kinase gene (TK gene) nanopartides and to investigate the expression of TK gene.METHODS: Poly(D,L-lactic-co-glycolic acid) (PLGA), a biodegradable and biocompatible polymer, was used to prepar...AIM:To prepare thymidine kinase gene (TK gene) nanopartides and to investigate the expression of TK gene.METHODS: Poly(D,L-lactic-co-glycolic acid) (PLGA), a biodegradable and biocompatible polymer, was used to prepare recombinant plasmid p^EGFP-AFP nanoparticles by a double-emulsion evaporation technique. Characteristics of the nanoparticles were investigated in this study, including morphology, entrapment efficiency, and tissue distribution.The expression of TK gene was also investigated by MTT assay, by which the viable cells were determined alter the addition of ganciclovir (GCV).The enhanced green fluorescent protein (EGFP) expression in human hepatocellular carcinoma SMMC-7721 cells and normal parenchymal Chang liver cellswere assessed by flow cytometry.RESULTS: The prepared plasmid-nanoparticles had regular spherical surface and narrow particle size span with a mean diameter of 72±12nm.The mean entrapment efficiency was 91.25%. A total of 80.14% DNA was found to be localized in the livers after 1-h injection with ^32P-DNA-PLGA nanoparticles in mouse caudal vein. The expression of DNA encapsulated in nanopartides was much higher than that in naked DNA, and human hepatocellular carcinoma SMMC-7721 cells were more sensitive to GCV than human normal parenchymal Chang liver cells.CONCLUSION:The enhanced transfection efficiency and stronger ability to protect plasmid DNA from being degraded by nucleases are due to nanoparticles encapsulation.展开更多
Aim To develop a sensitive competitive ELISA for the determination of biotin in transformed yeast culture media.Methods The ELISA plate was firstly coated with Mycoplasma hyopneumoniae, and then successively incubated...Aim To develop a sensitive competitive ELISA for the determination of biotin in transformed yeast culture media.Methods The ELISA plate was firstly coated with Mycoplasma hyopneumoniae, and then successively incubated with rabbit ami-Mycoplasma hyopneumoniae serum and goat anti-rabbit IgG-biotin to form the solid biotin, which competed with the biotin in the solution (standard or sample) for the limited streptavidin-horse radish peroxidase conjugate. The standard calibration curve for biotin analysis was constructed in the range of 50-2000ng·L^-1. Results The detection limit for biotin was found to be 83 ng·L^-1 , which waa about 1000 times lower than the lowest determination concenlration in the reported ELISA for biotin analysis. The relative standard deviations for the spiked samples at biotin concerarations of 200 ng·L^-1, 500 ng·L^-1 , and 1000 ng·L^-1 were 24.87%, 6.15%, and 7.86%, respectively, with the average recovery of 101.13%. The wild yeast and its sixty-three transformed yeast culture media were applied to the developed ELISA for the determination of biotin. It was found that the biotin concentrations in more than 85 % of the tested samples were enhanced with different increase factors after transformation. Conclusion Utilization of Mycoplasma hyopnetunoniae as the coating protein improves the precision and accmacy oftbe ELISA assay, which might be used for the biotin assay in other media.展开更多
The enzymatic synthesis of CCK-8 tripeptide derivative Phac-Met-Asp(OMe)-Phe-NH2 is reported. Starting with Phac-Met-OCam, we have successfully synthesized the target tripeptide with three free or immobilized enzymes...The enzymatic synthesis of CCK-8 tripeptide derivative Phac-Met-Asp(OMe)-Phe-NH2 is reported. Starting with Phac-Met-OCam, we have successfully synthesized the target tripeptide with three free or immobilized enzymes, ?chymotrypsin, papain and thermolysin in reasonable yields. The key steps in this synthesis were the coupling of Phac-Met-OCam and H-Asp(OMe)2 to form Met-Asp peptide bond catalyzed by ?chymotrypsin and the selective hydrolysis of -ester of Phac-Met-Asp(OMe)2 catalyzed by papain.展开更多
The synthesis of CCK - 4 (H - Trp- Met- Asp- Phe- NH2 ) by using enzym es exclusively was described.As protection group for the amino group we used the Phenylacetyl group (Phac) which had been cleaved at the end of ...The synthesis of CCK - 4 (H - Trp- Met- Asp- Phe- NH2 ) by using enzym es exclusively was described.As protection group for the amino group we used the Phenylacetyl group (Phac) which had been cleaved at the end of the synthesis with Penicillin G Amidase (PGA ) without affecting the peptide bonds.Thus,beginning with Phac- Trp- OH we had successfully synthesized the target peptide with following4 enzymes,α- Chym otrypsin,Papain,Therm olysin and PGA in four reac- tion steps.All reactions were carried out in aqueous buffer in reasonable yields(>6 5 % ) .FAB- MS or FD- MS verified the correct molecular mass of all peptides.展开更多
基金Supported by the National Natural Science Foundation of China,No. 30271613
文摘AIM: To investigate the uptake difference between bovine serum albumin nanoparticle (BSA-NP) and bovine serum albumin nanoparticles with their surface modified byglycyrrhizin (BSA-NP-GL) and to develop a novel hepatocyte targeting BSA-NP-GL based on active targeting technology mediated by specific binding site of GL on rat cellular membrane. METHODS: Calcein loaded bovine serum albumin nanoparticles (Cal-BSA-NP) were prepared by desolvation process. Glycyrrhizin was conjugated to the surface reactive amino groups (SRAG) of Cal-BSA-NP by sodium periodate oxidization, which resulted in calcein-loaded bovine serum albumin nanoparticles with their surface modified by glycyrrhizin (Cal-BSA-NP-GL). The morphology of the two types of prepared nanoparticles (NP) was observed by transmission electron microscopy. The diameter of NP was measured with a laser particle size analyzer. The interaction between Cal-BSA-NP-GL and primary cultured hepatocytes was studied through cellular uptake experiments. The uptake amount of Cal-BSA-NPGL and Cal-BSA-NP by rat hepatocytes was determinedby fluorospectrophotometry. Uptake characteristics were investigated through experiments of competitive inhibition of specific binding site of GL. RESULTS: Both Cal-BSA-NP-GL and Cal-BSA-NP had regular spherical surfaces. The average diameter of CalBSA-NP-GL and Cal-BSA-NP was 77 and 79 nm respectively. The uptake amount of the two NP by hepatocytes reached its maximum at 2 h after incubation. The uptake amount of Cal-BSA-NP-GL by rat hepatocytes was 4.43-fold higher than that of Cal-BSA-NP. There was a significant difference in the uptake of Cal-BSA-NP-GL and Cal-BSA-NP by hepatocytes (P<0.01). The uptake of Cal-BSA-NP-GL was inhibited when GL was added previously to isolated rat hepatocytes, and the uptake of Cal-BSA-NP was not affected by GL.CONCLUSION: A binding site of GL is present on the surface of rat hepatocytes, BSA-NP-GL may be internalized via this site by hepatocytes and can be used as a drug carrier for active targeting of delivery drugs to hepatocytes.
基金Supported by the National High Technology Research and Development Program of China(863 program),No.2001AA218021
文摘AIM:To develop a novel non-viral gene delivery system,which has a small particle size and a high transfection efficiency to hepatocyte and hepatoma cells.METHODS:Lipid-polycation-DNA lipopolyplex (LPD) was prepared by mixing plasmid DNA and polylysine. The resulted polyplex was incubated for 10min at room temperature,following the addition of preformed cationic liposomes. The morphology of LPD was observed by transmission electron microscopy.The diameter and surface charge of LPD were measured by photon correlation spectroscopy (PCS). The nuclease protection ability of LPD was evaluated by agarose gel electrophoresis. Estimation of the transfection efficiency was performed by galactosidase assay in Chang cells and SMMC-7721 cells.RESULTS:LPD had a regular spherical surface. The average diameter and the zeta potential of LPD were 132.1nm and 26.8mV respectively. LPD could protect plasmid DNA from nuclease degradation after 2 hours incubation at 37℃ while the naked DNA degraded rapidly. The average transfection efficiencies were 86.2±8.9% and 72.4±6.5% in Chang cells and SMMC-7721 cells respectively.CONCLUSION: LPD has a rather small particle size and a high transfection activity. LPD may be a good non-viral vector for application in some gene delivery.
文摘AIM:To prepare thymidine kinase gene (TK gene) nanopartides and to investigate the expression of TK gene.METHODS: Poly(D,L-lactic-co-glycolic acid) (PLGA), a biodegradable and biocompatible polymer, was used to prepare recombinant plasmid p^EGFP-AFP nanoparticles by a double-emulsion evaporation technique. Characteristics of the nanoparticles were investigated in this study, including morphology, entrapment efficiency, and tissue distribution.The expression of TK gene was also investigated by MTT assay, by which the viable cells were determined alter the addition of ganciclovir (GCV).The enhanced green fluorescent protein (EGFP) expression in human hepatocellular carcinoma SMMC-7721 cells and normal parenchymal Chang liver cellswere assessed by flow cytometry.RESULTS: The prepared plasmid-nanoparticles had regular spherical surface and narrow particle size span with a mean diameter of 72±12nm.The mean entrapment efficiency was 91.25%. A total of 80.14% DNA was found to be localized in the livers after 1-h injection with ^32P-DNA-PLGA nanoparticles in mouse caudal vein. The expression of DNA encapsulated in nanopartides was much higher than that in naked DNA, and human hepatocellular carcinoma SMMC-7721 cells were more sensitive to GCV than human normal parenchymal Chang liver cells.CONCLUSION:The enhanced transfection efficiency and stronger ability to protect plasmid DNA from being degraded by nucleases are due to nanoparticles encapsulation.
文摘Aim To develop a sensitive competitive ELISA for the determination of biotin in transformed yeast culture media.Methods The ELISA plate was firstly coated with Mycoplasma hyopneumoniae, and then successively incubated with rabbit ami-Mycoplasma hyopneumoniae serum and goat anti-rabbit IgG-biotin to form the solid biotin, which competed with the biotin in the solution (standard or sample) for the limited streptavidin-horse radish peroxidase conjugate. The standard calibration curve for biotin analysis was constructed in the range of 50-2000ng·L^-1. Results The detection limit for biotin was found to be 83 ng·L^-1 , which waa about 1000 times lower than the lowest determination concenlration in the reported ELISA for biotin analysis. The relative standard deviations for the spiked samples at biotin concerarations of 200 ng·L^-1, 500 ng·L^-1 , and 1000 ng·L^-1 were 24.87%, 6.15%, and 7.86%, respectively, with the average recovery of 101.13%. The wild yeast and its sixty-three transformed yeast culture media were applied to the developed ELISA for the determination of biotin. It was found that the biotin concentrations in more than 85 % of the tested samples were enhanced with different increase factors after transformation. Conclusion Utilization of Mycoplasma hyopnetunoniae as the coating protein improves the precision and accmacy oftbe ELISA assay, which might be used for the biotin assay in other media.
文摘The enzymatic synthesis of CCK-8 tripeptide derivative Phac-Met-Asp(OMe)-Phe-NH2 is reported. Starting with Phac-Met-OCam, we have successfully synthesized the target tripeptide with three free or immobilized enzymes, ?chymotrypsin, papain and thermolysin in reasonable yields. The key steps in this synthesis were the coupling of Phac-Met-OCam and H-Asp(OMe)2 to form Met-Asp peptide bond catalyzed by ?chymotrypsin and the selective hydrolysis of -ester of Phac-Met-Asp(OMe)2 catalyzed by papain.
文摘The synthesis of CCK - 4 (H - Trp- Met- Asp- Phe- NH2 ) by using enzym es exclusively was described.As protection group for the amino group we used the Phenylacetyl group (Phac) which had been cleaved at the end of the synthesis with Penicillin G Amidase (PGA ) without affecting the peptide bonds.Thus,beginning with Phac- Trp- OH we had successfully synthesized the target peptide with following4 enzymes,α- Chym otrypsin,Papain,Therm olysin and PGA in four reac- tion steps.All reactions were carried out in aqueous buffer in reasonable yields(>6 5 % ) .FAB- MS or FD- MS verified the correct molecular mass of all peptides.