Construction of serum-resistant cationic polymer α-CD-PAMAM and evaluation of its performances as gene delivery vector / 药学学报

Polyamidoamine (PAMAM) dendrimers as synthetic gene vectors are efficient gene delivery systems. In this study, a kind of α-cyclodextrin-PAMAM conjugates polymer (CyD-G1) was synthesized as a gene delivery vector. Based on 1H NMR detectation, about 6.4 PAMAM-G1 molecules was grafted onto an α-CD core. Agarose gel electrophoresis revealed that CyD-G1 could efficiently bind with DNA to condense them into nano-scale particles, which showed a similar binding capacity of PEI-25K. Besides, it could protect DNA from DNase I degradation in a low N/P ratio. When N/P ratio in the CyD-G1/DNA polyplex was 40, the average particle size of CyD-G1/DNA polyplex was about 120 nm, and zeta potential was +21 mV. This polyplex could maintain its particle size in serum-containing solution within 360 min. In comparison with PEI-25K carrier, CyD-G1 showed low cytotoxicity in various cell lines. Cell transfection results showed that CyD-G1 efficiently delivered DNA into cells at N/P=80 compared with Lipofectamine 2000 and PEI-25K.Unlike Lipofectamine 2000 and PEI-25K, in serum-containing test condition, CyD-G1/DNA polyplex could maintain the transgene activities. The results of confocal laser scanning microscopy indicated that most DNA entered into cell nuclei within 4 h, and this phenomenon was consistent with the results calculated by flow cytometry. Taken together, CyD-G1 showed good transgene activities and the gene delivery vector could be used not only in vitro but also in vivo.

PEGylation of polyamidoamine dendrimer and the properties for gene vectors / 药学学报

Polyamidoamine-polyethylene glycol (PAMAM-PEG) copolymers were synthesized using IPDI as coupling reagent by two-step method. The copolymers were characterized by IR spectrum and 1H NMR spectrum, and the PEG conjugating ratios of the copolymers were calculated equal to 10% and 30% separately. MTT assay indicated that after PEGylation a lower cytotoxicity of the copolymers could be found, and with increasing PEG conjugating ratio the cytotoxicity decreased obviously. Agarose gel retardation assay demonstrated that PAMAM-PEG copolymers could be combined with DNA and PAMAM-PEG/DNA complexes were prepared by self-assembly. DLS measurement showed that when N/P > or = 50, the particle size of copolymer/ gene complexes was in a range of 150-200 nm, and the zeta potential was in a range of 10-25 mV. In vitro gene transfection illustrated that when N/P < or = 50, the gene transfection efficiency of PAMAM-PEG copolymers was a little less than that of PAMAM-G5, but the transfection efficiency can be raised by increasing N/P ratio or transfection time. Considering both cytotoxicity and transfection efficiency aspects PAMAM-PEG-13 was more effect than PAMAM-PEG-39 in PEGylation.

In vitro study on polyethylene glycol-chitosan copolymer as a gene delivery vector / 药学学报

Chitosan and its derivatives are extensively studied as non-viral gene delivery vectors nowadays. Polyethylene glycol-chitosan (mPEG-CS) copolymers were synthesized by oxidation of mPEG-OH and then combined mPEG-CHO with amino groups on chitosan chains. The in vitro cytotoxicity of copolymers was evaluated by MTT method. The results showed > 70% cell viability of HeLa and A549 cells after incubation with mPEG-CS copolymer from concentration 5 to 100 microg x mL(-1). The mPEG-CS copolymers with various degrees of PEG substitution were combined with DNA and the properties of mPEG-CS/DNA complexes were investigated such as nanoparticle size, zeta potential and agarose gel analysis. The best one among all these mPEG-CS copolymers was mPEG (3.55) -CS, for its capability to condense plasmid DNA was most efficient. For this reason, mPEG (3.55) -CS was picked out to mediate plasmid enhanced green fluorescence protein (pEGFP) and transfect HeLa and A549 cells. The expression of green fluorescence protein was observed by fluorescence microscope and the transfection efficiency was detected by flow cytometry. The gene expression mediated by mPEG-CS was resistant to serum, and the optimal transfection efficiency (8.1% for HeLa cells and 4.8% for A549 cells) of mPEG-CS/EGFP system was obtained under the condition of N/P 40 and 48 h transfection time. These results indicate that mPEG-CS copolymer is an efficient non-viral gene vector.

Preparation and properties of self-assemble paclitaxel-loaded core-shell type nano-micelles / 药学学报

Polyethylene glycol-polybenzyl-L-glutamate copolymer (PEG-PBLG) was synthesized and paclitaxel-loaded core-shell type nano-micelles with amphiphilic copolymer PEG-PBLG was prepared by the dialysis method. The drug loading content and entrapment efficiency were determined by HPLC. The average size and its distribution were determined by dynamic light scattering method. The paclitaxel release rate in vitro from micelles was measured by HPLC. The cell cytotoxicity in vitro was observed with MTT assay. The anti-tumor activity of paclitaxel-loaded micelles were evaluated in tumor-inhibiting test of nude mice using human liver cancer HepG-2. The results indicated that paclitaxel could be entrapped in PEG-PBLG copolymer micelles and its size was in the range of 80-265 nm which increased with an increase in molecular weight of PBLG in copolymer; in vitro the paclitaxel could be released sustainably from the micelles. In high concentration of paclitaxel (>20 microg x mL(-1)) the paclitaxel-loaded PEG-PBLG micelles displayed much less cell cytotoxicity than paclitaxel injections with Cremophor EL (P<0.05); the tumor inhibiting activity of paclitaxel-loaded PEG-PBLG micelles was similar to that of paclitaxel injections with Cremophor EL in the same paclitaxel concentration. It was concluded that the paclitaxel-loaded PEG-PBLG micelles had more uniform size and size distribution, excellent drug sustainable-release behavior, less cytotoxicity, good anti-tumor activity similar to paclitaxel injections with Cremophor EL. So paclitaxel-loaded PEG-PBLG micelles would be a novel paclitaxel preparation in clinic for the treatment of tumor.

Fluid shear stress regulates secretion of tissue-type plasminogen activator in human endothelial progenitor cells / 中华心血管病杂志

<p><b>OBJECTIVE</b>In order to investigate the role of shear stress in the regulation of endothelial function, we assessed here effects of shear stress on tissue-type plasminogen activator in human endothelial progenitor cells (EPCs).</p><p><b>METHODS</b>The peripheral blood mononuclear cells were separated from healthy adult and inducted into EPCs, which were identified by double staining for the fluorescent labeled acetylated-LDL and lectin. EPCs were seeded on the small diameter artificial vessels, and then divided into four different experimental groups including stationary group, low-flow shear stress group (5 dyn/cm(2)), medium-flow shear stress group (15 dyn/cm(2)) and high-flow shear stress group (25 dyn/cm(2)). The levels of t-PA in EPC culture medium at 0 hour, 5 hours, 10 hours, 15 hours, 20 hours and 25 hours after culture were measured by enzyme-linked immunosorbent assay.</p><p><b>RESULTS</b>The peripheral blood mononuclear cells differentiated into EPCs after induction, which were positively labeled by fluorescent acetylated-LDL and lectin. Shear stress enhanced production of the t-PA by EPCs, which was paralleled to levels and times of shear stress.</p><p><b>CONCLUSIONS</b>Shear stress increases t-PA secretion by human EPCs, suggesting that shear stress not only regulates vascular endothelial function but also participates in the pathogenesis of arteriosclerosis.</p>