The preparation method, serum stability, efficiency of cellular uptake and apoptosis induction of the cell penetrating peptide TAT and cleavable PEG co-modified liposomes loaded with paclitaxel (C-TAT-Lipo) were investigated. The best preparation procedure was performed by orthogonal test based on single factor screening method. First, the paclitaxel (PTX)-loaded liposomes were prepared by filming-rehydration method, evaluated with entrapment efficiency and polydispersity index. The morphology of C-TAT-Lipo was characterized by transmission electron microscopy. Turbidity variations were monitored in the presence of fetal bovine serum (FBS) to evaluate the serum stability of the liposomes developed here. Next, the efficiency of cellular uptake of different Rho-PE-labeled liposomes on B16F1 cells in vitro was evaluated by confocal laser scanning microscopy (CLSM) and flow cytometry. The quantitative analysis of apoptosis induced by different PTX-loaded liposomes was performed by Annexin V-FITC/PI double staining. The optimal formulation was as follows: Chol : lipid: 1 : 8 (molar ratio); drug : lipid: 1 : 40 (mass ratio); lipid concentration: 3 mmol x L(-1); temperature of hydration: 25 degrees C. The mean size and polydispersity index of C-TAT-Lipo were about (97.97 +/- 3.68) nm and 0.196 +/- 0.037, the zeta potential was (-0.89 +/- 0.45) mV, the entrapment efficiency of paclitaxel was (90.16 +/- 1.53)%. The particle sizes did not exhibit significant variations in 50% FBS over 24 h at 37 degrees C. The efficiency of cellular uptake of the C-TAT-Lipo increased 1.40 fold following the cleavage of PEG. Apoptosis analysis showed 59.3% increase of the apoptosis and necrosis profile of C-TAT-Lipo after the detachment of PEG shells, which was markedly higher than that of N-TAT-LP with or without glutathione and SL, respectively. The results indicate that the C-TAT-Lipo is successfully prepared by filming-rehydration method and shows significant antitumor activities.
Animals ; Annexin A5 ; Apoptosis ; Cell Line, Tumor ; Cell-Penetrating Peptides ; pharmacology ; Fluorescein-5-isothiocyanate ; analogs & derivatives ; Liposomes ; chemistry ; Melanoma, Experimental ; Mice ; Microscopy, Confocal ; Paclitaxel ; pharmacology ; Particle Size ; Polyethylene Glycols ; chemistry

The aimed of this study was to prepare stabilized thiomers to overcome the poor stability character of traditional thiomers. Poly(acrylic acid)-cysteine (PAA-Cys) was synthesized by conjugating cysteine with poly(acrylic acid) and poly(acrylic acid)-cysteine-6-mercaptonicotinic acid (PAA-Cys-6MNA, stabilized thiomers) was synthesized by grafting a protecting group 6-mercaptonicotinic acid (6MNA) with PAA-Cys. The free thiol of PAA-Cys was determined by Ellmann's reagent method and the ratio of 6MNA coupled was determined by glutathione reduction method. The study of permeation enhancement and stabilized function was conducted by using Franz diffusion cell method, with fluorescein isothiocyanate dextran (FD4) used as model drug. The influence of polymers on tight junctions of Caco-2 cell monolayer was detected with laser scanning confocal fluorescence microscope. The results indicated that both PAA-Cys and PAA-Cys-6MNA could promote the permeation of FD4 across excised rat intestine, and the permeation function of PAA-Cys-6MNA was not influence by the pH of the storage environment and the oxidation of air after the protecting group 6MNA was grafted. The distribution of tight junction protein of Caco-2 cell monolayer F-actin was influenced after incubation with PAA-Cys and PAA-Cys-6MNA. In conclusion, stabilized thiomers (PAA-Cys-6MNA) maintained the permeation function compared with the traditional thiomers (PAA-Cys) and its stability was improved. The mechanism of the permeation enhancement function of the polymers might be related to their influence on tight junction relating proteins of cells.
Acrylic Resins ; chemistry ; Actins ; metabolism ; Animals ; Caco-2 Cells ; Cysteine ; chemistry ; Dextrans ; Fluorescein-5-isothiocyanate ; analogs & derivatives ; Glutathione ; Humans ; Intestinal Absorption ; Intestinal Mucosa ; drug effects ; Nicotinic Acids ; chemistry ; Rats ; Sulfhydryl Compounds ; chemistry

Astragalus polysaccharides was lounded to 4-(2-aminoethylphenol), followed by labeling the APS-Tyr with fluorescein-5-isothiocyanate (FITC) at the secondary amino group. The absorption enhancement effects of low molecular weight chitosan and protamine on astragalus polysaccharides were evaluated via Caco-2 cell culture model. The results show that the fluorecent labeling compound has good stability and high sensitivity. On the other hand low molecular weight chitosan and protamine also can promoted absorption of the astragalus polysaccharides without any cytotoxity, and the absorption increase was more significant with increasing the amount of low molecular weight chitosan and protamine. At the same time, the low molecular weight chitosan has slightly better effect. The transepithelial electric resistance (TEER) of Caco-2 cells show that absorption enhancers could improve its membrane transport permeability by opening tight junctions between cells and increasing the cell membrane fluidity.
Absorption ; Astragalus Plant ; chemistry ; Biological Transport ; Caco-2 Cells ; Fluorescein-5-isothiocyanate ; chemistry ; Fluorescent Dyes ; chemistry ; Humans ; Plant Extracts ; chemistry ; pharmacokinetics ; Polysaccharides ; chemistry ; pharmacokinetics

To evaluate the effect of chlorogenic acid (CGA), a polyphenol abundant in coffee, on retinal vascular leakage in the rat model of diabetic retinopathy, Sprague-Dawley rats were divided into four groups: controls, streptozotocin-induced diabetic rats, and diabetic rats treated with 10 and 20 mg/kg chlorogenic acid intraperitoneally daily for 14 days, respectively. Blood-retinal barrier (BRB) breakdown was evaluated using FITC-dextran. Vascular endothelial growth factor (VEGF) distribution and expression level was evaluated with immunohistochemistry and Western blot analysis. Expression of tight junction proteins, occludin and claudin-5, and zonula occludens protein, ZO-1 was also evaluated with immunohistochemistry and Western blot analysis. BRB breakdown and increased vascular leakage was found in diabetic rats, with increased VEGF expression and down-regulation of occludin, claudin-5, and ZO-1. CGA treatment effectively preserved the expression of occludin, and decreased VEGF levels, leading to less BRB breakdown and less vascular leakage. CGA may have a preventive role in BRB breakdown in diabetic retinopathy by preserving tight junction protein levels and low VEGF levels.
Animals ; Blood-Retinal Barrier/*drug effects ; Chlorogenic Acid/metabolism/*pharmacology ; Claudin-5/metabolism ; Dextrans/chemistry ; Diabetes Mellitus, Experimental/complications/metabolism/*pathology ; Diabetic Retinopathy/etiology/prevention & control ; Down-Regulation ; Fluorescein-5-isothiocyanate/chemistry ; Male ; Occludin/metabolism ; Rats ; Rats, Sprague-Dawley ; Retina/*metabolism ; Tight Junction Proteins/metabolism ; Vascular Endothelial Growth Factor A/metabolism ; Zonula Occludens-1 Protein/metabolism

BACKGROUND: We developed a single-color multitarget flow cytometry (SM-FC) assay, a single-tube assay with graded mean fluorescence intensities (MFIs). We evaluated the repeatability of SM-FC, and its correlation with multicolor flow cytometry (MFC), to assess its application as a routine FC assay. METHODS: We selected CD19, CD3, CD4, and CD8 as antigen targets to analyze a lymphocyte subset. MFIs were graded by adjusting monoclonal antibody (mAb) volumes to detect several cell populations. Dimly labeled mAb was prepared by decreasing mAb volume and the optimum diluted volume was determined by serial dilution. SM-FC repeatability was analyzed 10 times in 2 normal controls. The correlation between SM-FC and MFC was evaluated in 20 normal and 23 patient samples. RESULTS: CV values (0.8-5.0% and 1.3-4.1% in samples 1 and 2, respectively) acquired by SM-FC with CD3-fluorescein alpha-isothyocyanate (FITC)dim+CD4-FITCbright and with CD19-FITCdim+CD3-FITCbright showed good repeatability, comparable to that acquired by MFC (1.6-3.7% and 1.0-4.8% in samples 1 and 2, respectively). Excellent correlation was observed between the 2 methods in the 20 normal samples (B cells, T cells, non-Thelper cells, and Thelper cells; r2=0.87, 0.97, 0.97, and 0.98, respectively; P<0.05). There were also linear relationships between SM-FC with CD19-FITCdim+CD3-FITCbright and CD8-PEdim+CD4-PEbright, and MFC, in the 23 patient samples (B cells, T cells, Tcytotoxic cells, and Thelper cells; r2> or =0.98, 0.99, 0.99, and 0.99, respectively; P<0.05). CONCLUSIONS: The multicolor, single-tube SM-FC technique is a potential alternative tool for identifying a lymphocyte subset.
Antibodies, Monoclonal/chemistry/*immunology ; Antigens, CD19/chemistry/metabolism ; Antigens, CD3/chemistry/metabolism ; Antigens, CD4/chemistry/metabolism ; Antigens, CD8/chemistry/metabolism ; B-Lymphocyte Subsets/immunology/metabolism ; Color ; Flow Cytometry/*methods ; Fluorescein-5-isothiocyanate/*chemistry ; Humans ; T-Lymphocyte Subsets/immunology/metabolism

This study is to evaluate the sustained-release effect of the thermosensitive in situ gel for injection of boanmycin hydrochloride (BAM) by bioluminescence imaging in nude mice. BAM was labeled with fluorescein isothiocyanate (FITC). The FITC-labeled BAM (FITC-BAM) was purified by dialysis and Sephadex G25 gel column, and then was identified by matrix-assisted laser desorption ionization/time of flight (MALDI-TOF). The model of experimental hepatoma HepG-2 nude mice was established, and the optical imaging system was applied to evaluate the distribution of FITC-BAM in vivo. Results of MALDI-TOF proved that the major molecular ratio of BAM : FITC was 1 : 1 or 1 : 2. Bioluminescence imaging showed that the diffusion of FITC-BAM in situ gel group was significantly delayed compared with the negative control group. This study demonstrated that the thermosensitive in situ gel can effectively delay the release of boanmycin hydrochloride, and extend the retention time in vivo.
Animals ; Antibiotics, Antineoplastic ; administration & dosage ; chemistry ; pharmacokinetics ; Bleomycin ; administration & dosage ; analogs & derivatives ; chemistry ; pharmacokinetics ; Delayed-Action Preparations ; Drug Carriers ; chemistry ; Female ; Fluorescein-5-isothiocyanate ; administration & dosage ; chemistry ; pharmacokinetics ; Gels ; chemistry ; Hep G2 Cells ; Humans ; Injections ; Mice ; Mice, Inbred BALB C ; Mice, Nude ; Neoplasm Transplantation ; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization ; Temperature ; Tissue Distribution ; Viscosity

BACKGROUND: Since the recent introduction of radioimmunotherapy (RIT) using antibodies against cluster of differentiation (CD) 45 for the treatment of lymphoma, the clinical significance of the CD45 antigen has been increasing steadily. Here, we analyzed CD45 expression on lymphocyte subsets using flow cytometry in order to predict the susceptibility of normal lymphocytes to RIT. METHODS: Peripheral blood specimens were collected from 14 healthy individuals aged 25-54 yr. The mean fluorescence intensity (MFI) of the cell surface antigens was measured using a FACSCanto II system (Becton Dickinson Bioscience, USA). MFI values were converted into antibody binding capacity values using a Quantum Simply Cellular microbead kit (Bangs Laboratories, Inc., USA). RESULTS: Among the lymphocyte subsets, the expression of CD45 was the highest (725,368+/-42,763) on natural killer T (NKT) cells, 674,030+/-48,187 on cytotoxic/suppressor T cells, 588,750+/-48,090 on natural killer (NK) cells, 580,211+/-29,168 on helper T (Th) cells, and 499,436+/-21,737 on B cells. The Th cells and NK cells expressed a similar level of CD45 (P=0.502). Forward scatter was the highest in NKT cells (P<0.05), whereas side scatter differed significantly between each of the lymphocyte subsets (P<0.05). CD3 expression was highest in the Th and NKT cells. CONCLUSIONS: NKT cells express the highest levels of CD45 antigen. Therefore, this lymphocyte subset would be most profoundly affected by RIT or pretargeted RIT. The monitoring of this lymphocyte subset during and after RIT should prove helpful.
Adult ; Antibodies/immunology ; Antigens, CD45/*analysis/immunology ; B-Lymphocytes/immunology/metabolism ; CD8-Positive T-Lymphocytes/immunology/metabolism ; Female ; Flow Cytometry/*methods ; Fluorescein-5-isothiocyanate/chemistry ; Fluorescent Dyes/chemistry ; Humans ; Killer Cells, Natural/immunology/metabolism ; Lymphocytes/immunology/*metabolism ; Lymphoma/radiotherapy ; Male ; Middle Aged ; Natural Killer T-Cells/immunology/metabolism ; Protein Binding ; Radioimmunotherapy ; Reagent Kits, Diagnostic ; T-Lymphocytes, Helper-Inducer/immunology/metabolism

BACKGROUND: Since the recent introduction of radioimmunotherapy (RIT) using antibodies against cluster of differentiation (CD) 45 for the treatment of lymphoma, the clinical significance of the CD45 antigen has been increasing steadily. Here, we analyzed CD45 expression on lymphocyte subsets using flow cytometry in order to predict the susceptibility of normal lymphocytes to RIT. METHODS: Peripheral blood specimens were collected from 14 healthy individuals aged 25-54 yr. The mean fluorescence intensity (MFI) of the cell surface antigens was measured using a FACSCanto II system (Becton Dickinson Bioscience, USA). MFI values were converted into antibody binding capacity values using a Quantum Simply Cellular microbead kit (Bangs Laboratories, Inc., USA). RESULTS: Among the lymphocyte subsets, the expression of CD45 was the highest (725,368+/-42,763) on natural killer T (NKT) cells, 674,030+/-48,187 on cytotoxic/suppressor T cells, 588,750+/-48,090 on natural killer (NK) cells, 580,211+/-29,168 on helper T (Th) cells, and 499,436+/-21,737 on B cells. The Th cells and NK cells expressed a similar level of CD45 (P=0.502). Forward scatter was the highest in NKT cells (P<0.05), whereas side scatter differed significantly between each of the lymphocyte subsets (P<0.05). CD3 expression was highest in the Th and NKT cells. CONCLUSIONS: NKT cells express the highest levels of CD45 antigen. Therefore, this lymphocyte subset would be most profoundly affected by RIT or pretargeted RIT. The monitoring of this lymphocyte subset during and after RIT should prove helpful.
Adult ; Antibodies/immunology ; Antigens, CD45/*analysis/immunology ; B-Lymphocytes/immunology/metabolism ; CD8-Positive T-Lymphocytes/immunology/metabolism ; Female ; Flow Cytometry/*methods ; Fluorescein-5-isothiocyanate/chemistry ; Fluorescent Dyes/chemistry ; Humans ; Killer Cells, Natural/immunology/metabolism ; Lymphocytes/immunology/*metabolism ; Lymphoma/radiotherapy ; Male ; Middle Aged ; Natural Killer T-Cells/immunology/metabolism ; Protein Binding ; Radioimmunotherapy ; Reagent Kits, Diagnostic ; T-Lymphocytes, Helper-Inducer/immunology/metabolism

An in vitro detection method of the gastrointestinal absorption of Pilose Antler protein was established for mixed protein activity. Five bands of protein with molecular weight of 17.8-160 kD derived from the Pilose Antler were extracted and sufficiently labeled with FITC (FITC-PE). The stability and variation of FITC-PE in gastrointestinal circumstances were detected by native polyacrylamide gel electrophoresis and confocal laser scanning microscope. Results showed that the main component of FITC-PE kept invariant after being reacted with artificial gastric fluid and artificial intestinal fluid. The fluorescence signal was detected 20 min after administration in the valgus intestinal purse experiment, and three kinds of protein, with molecular weight of 45, 25, and 17.8 kD, were detected in the mixture of absorbent protein. The research laid the foundation for the further in vivo study of Pilose Antler protein. Meanwhile, it would be an in vitro screening method for the absorption, distribution and metabolism of mixed protein from traditional Chinese medicine.
Animals ; Antlers ; chemistry ; Deer ; Fluorescein-5-isothiocyanate ; Intestinal Absorption ; Intestinal Mucosa ; metabolism ; Male ; Materia Medica ; chemistry ; isolation & purification ; pharmacokinetics ; Microscopy, Confocal ; Molecular Weight ; Native Polyacrylamide Gel Electrophoresis ; Proteins ; chemistry ; isolation & purification ; pharmacokinetics ; Rats ; Rats, Wistar ; Stomach ; metabolism

The lipase labeled with the fluorescein isothiocyanat (FITC) was immobilized on the derivatives of the polyethylene glycol. The article discussed the effect of factors on the characters of lipase and analyzed the relationships among the activity of lipase, conformation, and fluorescence spectrum while the activity and the fluorescence spectrum of immobilized lipase were determined. The results demonstrated that polyethylene glycol 400-diacrylate could form appropriate network to improve the activity of enzyme. Adding ligand induced the lipase's catalytic conformation to increase the activity twice more than before. The active centre of lipase could be released by the extraction of ligand thus increasing the activity. After immobilization, the stability of labeled lipase improved greatly: immobilized lipases retained more than 70% and 60% of initial activity under conditions of 90 degrees C and strong acid or alkali, respectively. After immersing immobilized lipases into guanidine hydrochloride or urea for 15 days, the lipases retained upwards of 70% activity. The fluorescence spectrum could obviously reflect the changes of the activity and conformation of lipase. The fluorescence intensity was the minimum in the optimal pH and temperature. In the denaturing agent it declined as time passed. These results indicated that the unfolded processes of immobilized lipases are different under different conditions.
Dextrans ; chemistry ; Enzyme Stability ; Enzymes, Immobilized ; chemistry ; metabolism ; Fluorescein-5-isothiocyanate ; analogs & derivatives ; chemistry ; Fluorescent Dyes ; chemistry ; Lipase ; chemistry ; metabolism ; Polyethylene Glycols ; chemistry ; Protein Unfolding ; drug effects