[Effect of functionalized multi-walled carbon nanotubes on L02 cells].

OBJECTIVE: To investigate the cytotoxic effect of multi-walled carbon nanotubes (MWCNTs) on human liver L02 cells and its relevant mechanism. METHODS: MWCNTs, carboxyl modification MWCNTs (MWCNTs-COOH), and hydroxyl modification MWCNTs (MWCNTs-OH) were characterized by transmission electron microscopy, scanning electron microscopy, and X-ray photoelectron spectroscopy. The carbon nanotubes at concentrations of 12.5, 25, 50, 100, and 200 µg/ml were incubated with human liver L02 cells for 24, 48 and 72 hours, respectively. The cell viability was evaluated by water soluble tetrazolium salts assay and the intercellular reactive oxygen species induced by the carbon nanotubes were detected by 2', 7'-dichlorodihydrofluorescein diacetate method. RESULTS: Transmission electron microscope showed that the average outside diameters (10 to 20 nm) and the average length (10 to 30 µm) of the three MWCNTs were similar. Scanning electron microscope indicated that the three MWCNTs had a similar surface topography. X-ray photoelectron spectroscopy demonstrated that the MWCNTs-COOH and MWCNTs-OH had relatively high peak areas at 289 and 286ev, respectively,indicating that they have been modified by carboxyl and hydroxyl groups,respectively. Water soluble tetrazolium salts assay showed that the MWCNTs-COOH was less cytotoxic when compared to MWCNTs which demonstrated to be slightly more cytotoxic than MWCNTs-OH. The capability to induce increase in intracellular reactive oxygen species was in the following order: MWCNTs > MWCNTs-COOH > MWCNTs-OH. CONCLUSIONS: Modification of MWCNTs with carboxyl group and hydroxyl group improves the biocompatibility of MWCNTs to some extents. MWCNTs-COOH has better compatibility than MWCNTs at the low concentration,and MWCNTs-OH showed better compatibility than MWCNTs after 48 hours. Different mechanisms may be involved in the interaction between cells and the MWCNTs with different chemical surfaces.

[Effect of chitosan gene nanoparticles on L02 cells].

OBJECTIVE: To investigate the effect of the gene nanoparticles using chitosan (CNP), arginine modified chitosan (ANP), or hexadecylated chitosan (HNP) as carriers on the human normal liver cell line L02. METHODS: CNPs, ANPs, and HNPs were prepared using complex coacervation method. The size and zeta potential of the gene nanoparticles were measured using Zetasizer nanoZS. The nanoparticles at concentrations of 5, 10, 30, and 50 microg/ml (based on the content of DNA) were incubated with L02 cells, respectively. The cell viability was evaluated by MTT assay, and the effect of the gene nanoparticles on the cell apoptosis was analyzed by flow cytometry. RESULTS: The zeta potential of the gene nanoparticles ranged from 12.10 to 14.63 mV, and their diameters ranged from 148.07 to 179.47 nm. MTT assay showed that the viability of L02 cells began to decrease when the concentration of CNPs reached 30 microg/ml and higher. Furthermore, the CNPs could induce cell apoptosis as the concentration of CNPs reached 30 microg/ml and higher. CONCLUSION: CNPs can induce L02 cell apoptosis at relatively higher concentrations.

[Expression and purification of tissue factor pathway inhibitor in Pichia pastoris].

OBJECTIVE: To generate recombinant human tissue factor pathway inhibitor (TFPI) in Pichia pastoris. METHODS: To improve the expression of TFPI, a silent mutation was generated at the specific site of TFPI cDNA. Both wild-type TFPI cDNA and mutated TFPI cDNA were cloned into the expression vector pPic9. The constructed plasmids were subsequently transformed into Pichia pastoris cells GS115 and KM71, and the transformants were confirmed by polymerase chain reaction and DNA sequencing. The expression of recombinant protein was induced by addition of 0.5% methanol in the culture medium. The cell culture medium after induction was concentrated through ultra filtration. The recombinant protein was further purified by a three-step process (Heparin-sepharose CL-6B affinity chromatography, DEAE-Sepharose Fast Flow affinity chromatography, and Sephadex G75-gel filtration). The amount of the recombinant protein was quantified with gel imaging system. The activity of the recombinant protein was analyzed by the chromogenic substrate assay. RESULTS: The amount of TFPI expressed in the mutated clone (1 mg/L) was much higher than that in the wild type clone (0.1 mg/L). The TFPI activity in the recombinant GS115 cells could be detected 12 hours after induction and reached the peak at 36 hours, while the TFPI activity in the recombinant KM71 cells started to show up at 24 hours after induction and reached the peak at 72 hours. The expression of recombinant protein in the silent mutant was significantly higher than those of wild type clone in both GS115 and KM71 host cells. The relative molecular mass of recombinant TFPI was approximately 42 000. CONCLUSION: Introduction of the silent mutation at the specific site of TFPI cDNA can increase the recombinant protein expression in Pichia pastoris, which is much higher than that in insect cells or saccharomyces cerevisiae.

[A novel anti-DNA antibody modified coronary stent for site-specific plasmid DNA delivery].

OBJECTIVE: To explore the feasibility of using an endovascular metal stent as a highly efficient and site-specific gene delivery system. METHODS: Stents were formulated with a collagen coating. Anti-DNA monoclonal antibodies were covalently bound to the collagen surface by a cross linking reagent of N-succinimidyl 3-(2-pyridyldithio) propionate (SPDP). Binding capacity and stability of antibody and plasmid DNA on stents were quantified by radioactive labeling. The gene transduction efficiency was evaluated in cell culture and in rabbits. RESULTS: The amount of antibodies binding on collagen matrix through SPDP reaction was 15 times higher than that of through physical absorption (P < 0.005). The binding stability of plasmid was significantly better than the control groups (P < 0.01). There was no harmful effect on cell growth with the anti-DNA antibody modified stents. The stents retrieved from cell culture after 72 hours of incubation in A10 cells showed numerous transducted cells only infiltrating the surface coating indicating a highly localized and efficient gene delivery pattern. Results of in vivo gene transfer by this modified stent revealed (2.8 +/- 0.7)% of total cells transduction and the higher transduction location was neointimal layer (about 7%). No distal spread of vector was detectable in the anti-DNA antibody modified stent implantation animals. CONCLUSIONS: Anti-DNA antibody modified stents represent a novel highly efficient and site-specific gene delivery system which can deliver various kinds of plasmid vectors. The release of plasmid DNA tethered on the stents could be controlled in some conditions. This novel system provided a novel platform for cardiovascular site-specific gene therapy.

[Cellular uptake and cytotoxicity of modified chitosans as gene carriers].

OBJECTIVE: To evaluate the effects of arginine modified chitosan or hexadecylated modified chitosan as gene carriers on the cellular uptake by vascular smooth muscle cells and its in vitro cytotoxicity. METHODS Plasmid DNA was labeled with alpha-32P-dATP and complexed with the modified chitosans or unmodified chitosan to form nanoparticle complexes by complex coacervation method. Uptake of all kinds of chitosan/ DNA nanoparticle complexes (CNC) by A10 cells was measured by beta-liquid scintillation counting. The in vitro cytotoxicity of the CNC was evaluated by the 3-[4,5-dimethylthiazol-2-yl]-2, 5-diphenyltetrazolium bromide (MTT) assay. RESULTS: The diameters of the CNC ranged from 55.9-174.9 nm and the zeta potentials were from 10. 8 mV for the arginine modified chitosan/DNA nanoparticle complexes (ACNC) to 1.8 mV for the hexadecylated chitosan/DNA nanoparticle complexes (HCNC). The cellular uptake of the modified chitosan/ DNA nanoparticle complexes (MCNC) by A10 cells increased significantly when compared with the unmodified chitosan/DNA nanoparticle complexes (UCNC) (P < 0.05), with the HCNC at N/P ratio of 1:1 and the ACNC at ratio of 8:1 showing the highest cellular uptake (1.3 fold higher than UCNC, P < 0.05). MCNC were much less cytotoxic when compared with Lipofectamine 2000-DNA nanoparticles. CONCLUSION: DNA nanoparticle complexes prepared with either arginine or hexadecylated modified chitosan can improve the cellular uptake of the DNA, while the in vitro cytotoxicity of both of the modified chitosan is much less than that of Lipofectamine 2000.

Changes of chymase, angiotensin converting enzyme and angiotensin II type 1 receptor expressions in the hamster heart during the development of heart failure.

BACKGROUND: Little is known about the role of dual angiotensin II forming pathways during heart failure. In the present study, the changes of chymase and angiotensin converting enzyme (ACE) expressions in the failing hearts of hamsters were analysed. METHODS: Heart failure was induced by ligation of left anterior descending branch of the coronary artery. Chymase, ACE and angiotensin II type 1 receptor (AT1R) mRNA levels were analysed by reverse transcription polymerase chain reaction (RT-PCR). The activities of chymase and ACE were determined by radioimmunoassay (RIA). Myocardial collagen fibre analysis was performed under optical microscope. RESULTS: Left ventricular systolic pressure (LVSP) and maximum left ventricular developed pressure increase rate (dp/dtmax, mmHg/s) gradually moved lower at 2, 3, 4 and 8 weeks after operation. On the other hand, left ventricular end-diastolic pressure (LVEDP) increased gradually after operation. Compared with the control group (3.55 +/- 0.06, 4.79 +/- 0.70), the heart weight/body weight ratio in operation group had increased significantly at 4 weeks and 8 weeks (4.28 +/- 0.43, 6.17 +/- 0.73) (P < 0.01). Collagen staining showed that the quantity of myocardial collagen fibre increased significantly in the operation group. RT-PCR showed that the chymase mRNA level in the operation group was consistently greater than that in the control group. AT1R mRNA level was also increased significantly at 3 weeks and 4 weeks, both being 1.3 times that of the control group (P < 0.01), whereas ACE mRNA level was not changed. Higher activity of chymase was detected in operation group, being 4, 8, 13 and 19 times that of the control group at 2, 3, 4 and 8 weeks (P < 0.01), respectively. ACE activity was also significantly higher at the same time, being 7, 10, 10 and 3.5 times that of the control (P < 0.01). Angiotensin II (Ang II) level in operation group increased significantly, being 2.5, 2.7, 3.5 and 2 times that of the control group at 2, 3, 4 and 8 weeks, respectively (P < 0.01). CONCLUSIONS: A dual Ang II forming pathway from both ACE and chymase in the hamster hearts plays an important role during the development of heart failure. At the decompensatory stage, the reduction of AngII level may be associated with the decrease of ACE activity.

[Chemical coupling of anti-dNA antibody on collagen coating].

OBJECTIVE: To evaluate the feasibility and stability of chemically conjugating IgM on collagen films. METHODS: IgM was labeled with 125I using the chloramine-T method. Six collagen films were randomly divided into two groups. In chemical coupling group 125I-labeled IgM was chemically coupled with the films through N-succinmiclyl-3- (2-pyridyl-dithio) propionate reaction. In control group 125I-labeled IgM was absorbed onto collagen films. The amount of IgM on the collagen films and the amount of IgM remained on the films after extensive rinsing with phosphate buffered saline were monitored by counting the radioactivity of 125I. RESULTS: The amount of antibodies loaded onto collagen films in the chemical coupling group was 15 times higher than that on the control films, showing significant statistical difference (P < 0.01). And the stability of conjugation antibodies on collagen films was significantly better than the control films. CONCLUSION: Chemical coupling is an effective approach to immobilize antibodies on collagen for further plasmid DNA tethering.

Impact of the tissue factor pathway inhibitor gene on apoptosis in human vascular smooth muscle cells.

Tissue factor pathway inhibitor (TFPI) plays a vitally important role in the blood coagulation pathway. Recent studies indicated that TFPI induces apoptosis in vascular smooth-muscle cells (VSMCs) in animals. The present study investigated whether the TFPI gene could also induce apoptosis in human vascular smooth-muscle cells (hVSMCs). Such cells were isolated from human umbilical arteries and subsequently transfected with pIRES-TFPI plasmid (2 µg/mL). MTT assaying and cell counting were applied to measure cell viability and proliferation, RT-PCR was utilized to analyze TFPI gene expression in the cells. Apoptosis was analyzed by fluorescence activated cell sorting (FACS). Several key proteins involved in apoptosis were examined through Western blotting. It was shown that TFPI gene transfer led to its increased cellular expression, with a subsequent reduction in hVSMC proliferation. Further investigation demonstrated that TFPI gene expression resulted in lesser amounts of procaspase-3, procaspase-8 and procascase-9, and an increased release of mitochondrial cytochrome c (cyt-c) into cytoplasm, thereby implying the involvement of both extrinsic and intrinsic pathways in TFPI gene-induced apoptosis in hVSMCs.

Impact of the tissue factor pathway inhibitor gene on apoptosis in human vascular smooth muscle cells

Tissue factor pathway inhibitor (TFPI) plays a vitally important role in the blood coagulation pathway. Recent studies indicated that TFPI induces apoptosis in vascular smooth-muscle cells (VSMCs) in animals. The present study investigated whether the TFPI gene could also induce apoptosis in human vascular smooth-muscle cells (hVSMCs). Such cells were isolated from human umbilical arteries and subsequently transfected with pIRES-TFPI plasmid (2 μg/mL). MTT assaying and cell counting were applied to measure cell viability and proliferation, RT-PCR was utilized to analyze TFPI gene expression in the cells. Apoptosis was analyzed by fluorescence activated cell sorting (FACS). Several key proteins involved in apoptosis were examined through Western blotting. It was shown that TFPI gene transfer led to its increased cellular expression, with a subsequent reduction in hVSMC proliferation. Further investigation demonstrated that TFPI gene expression resulted in lesser amounts of procaspase-3, procaspase-8 and procascase-9, and an increased release of mitochondrial cytochrome c (cyt-c) into cytoplasm, thereby implying the involvement of both extrinsic and intrinsic pathways in TFPI gene-induced apoptosis in hVSMCs.