Large-scale production of lipoplexes with long shelf-life.

The instability of lipoplex formulations is a major obstacle to overcome before their commercial application in gene therapy. In this study, a continuous mixing technique for the large-scale preparation of lipoplexes followed by lyophilisation for increased stability and shelf-life has been developed. Lipoplexes were analysed for transfection efficiency and cytotoxicity in human aorta smooth muscle cells (HASMC) and a rat smooth muscle cell line (A-10 SMC). Homogeneity of lipid/DNA-products was investigated by photon correlation spectroscopy (PCS) and cryotransmission electron microscopy (cryo-TEM). Studies have been undertaken with DAC-30, a composition of 3beta-[N-(N,N'-dimethylaminoethane)-carbamoyl]-cholesterol (DAC-Chol) and dioleylphosphatidylethanolamine (DOPE) and a green fluorescent protein (GFP) expressing marker plasmid. A continuous mixing technique was compared to the small-scale preparation of lipoplexes by pipetting. Individual steps of the continuous mixing process were evaluated in order to optimise the manufacturing technique: lipid/plasmid ratio, composition of transfection medium, pre-treatment of the lipid, size of the mixing device, mixing procedure and the influence of the lyophilisation process. It could be shown that the method developed for production of lipoplexes on a large scale under sterile conditions led to lipoplexes with good transfection efficiencies combined with low cytotoxicity, improved characteristics and long shelf-life.     

Synergism in gene delivery by small PEIs and three different nonviral vectors

We have reported earlier that a combination of low-molecular weight polyethylenimines (PEIs) with the cationic liposome, Dosper, results in a synergistic increase in the transfection efficiency. Now we have investigated whether this synergism is a general mechanism seen with other transfection reagents as well. Therefore, we have combined the low-molecular weight PEIs (MW 700 and 2000) with Dotap (a monocationic liposome), Lipofectamine (a combination of neutral and polycationic liposome), and Superfect (a dendrimer). The highest synergism was achieved with Lipofectamine and PEIs in the SMC cells, or with Dotap and PEIs in the C6 cells. Superfect did not induce any synergism. The combinations did not cause any changes in DNA condensing ability measured with ethidium bromide exclusions. The proton pump inhibitor, bafilomycin A1, had similar effects in both cell lines. Interestingly, the combination of Dosper (a positive control) and PEI caused the most effective transfection synergism in the presence of serum, although Lipofectamine, with or without PEIs, was a very potent reagent demonstrating the best transfection efficiency in the absence of serum. It is suggested that the PEI/Dosper-mediated synergism in the transfection efficiency may be a general mechanism for liposomal transfection reagents, although the effects can vary depending on cell lines.     

三种阳离子转染试剂对小鼠血管瘤内皮细胞转染效率及细胞毒性的影响

目的 从三种常用的阳离子转染试剂中筛选出对小鼠血管瘤内皮细胞(EOMA)有较高转染效率和较低细胞毒性的转染试剂.方法 以含有增强型绿色荧光蛋白(EGFP)报告基因的真核表达载体pEGFP-N1为报告基因,以阳离子脂质体LipofectamineTM2000、LipofectamineTM PLUS和阳离子聚合物JetPEITM为转染试剂,按照转染试剂盒的说明优化其转染条件,分别转染EOMA细胞,24 h后在荧光显微镜下计数阳性细胞率、MTT法检测各转染条件下对EOMA的细胞毒性.结果 经优化转染条件,LipofectamineTM PLUS和JetPEITM转染的细胞中阳性细胞的最高比例分别为45%和47%,LipofectamineTM2000转染的细胞中荧光蛋白表达的比例最高(＞80%),Lipofecta-mineTM2000试剂在最高转染效率的剂量下仍然保证了80%的细胞存活率.结论 阳离子脂质体LipofectamineTM2000对小鼠EOMA细胞有较高的转染效率和较低的细胞毒作用.     

转染p21基因对人动脉平滑肌细胞生长的作用

目的探讨转染p21基因对人动脉平滑肌细胞（HASMC）的影响，以探讨转染p21基因实现抗冠状动脉支架内再狭窄的作用，为冠状动脉支架内再狭窄的治疗提供新思路。方法以Lipofectamine2000脂质体介导p21基因转染HASMC株；免疫组化法检测转染p21基因后，其编码蛋白在HASMCs的表达情况；描绘p21基因转染后HASMCs的增殖曲线；WST-1法测定OD值，计算细胞生长抑制率及流式细胞仪检测p21基因转染对HASMCs凋亡的影响。结果免疫组化法显示p21基因成功转入HASMCs后，其编码的蛋白能在细胞质内进行高表达；转染p21基因的HASMCs在体外生长曲线较对照组明显降低；WST-1法显示转染p21基因的HASMCs细胞活力与对照组相比有显著性差异（P〈0．01）；流式细胞仪检测发现转染p21基因的HASMCs细胞凋亡率达40％以上，且显著高于对照组。结论成功将p21基因转入HASMCs，其编码蛋白可能参与了抑制细胞生长和诱导细胞凋亡作用，提示p21基因转染HASMCs技术有可能为人冠状动脉支架内再狭窄的治疗提供一种新的防治策略与手段。     

转染p21基因对人动脉平滑肌细胞的影响

目的：探讨转染p21基因对人动脉平滑肌细胞（HASMC）的影响,以探讨转染p21基因实现抗冠状动脉支架内再狭窄的作用,为冠状动脉支架内再狭窄的治疗提供新思路。方法：以Lipo-fectamine 2000脂质体介导p21基因转染HASMC株;免疫组化法检测转染p21基因后,其编码蛋白在HASMCs的表达情况;描绘p21基因转染后HASMCs的增殖曲线;WST-1法测定OD值,计算细胞生长抑制率及流式细胞仪检测p21基因转染对HASMCs凋亡的影响。结果：免疫组化法显示p21基因成功转入HASMCs后,其编码的蛋白能在细胞核内进行高表达;转染p21基因的HASMCs在体外生长曲线较对照组明显降低;WST-1法显示转染p21基因的HASMCs细胞活力与对照组相比明显降低,差异有统计学意义（P〈0.05）;流式细胞仪检测发现转染p21基因的HASMCs细胞凋亡率达40.26%＋0.013%,且显著高于对照组,差异有统计学意义（P〈0.05）。结论：成功将p21基因转入HASMCs,其编码蛋白可能参与了抑制细胞生长和诱导细胞凋亡作用,提示p21基因转染HASMCs技术有可能为人冠状动脉支架内再狭窄的治疗提供一种新的防治策略与手段。     

Contribution of serum and cellular semicarbazide-sensitive amine oxidase to amine metabolism and cardiovascular toxicity.

Semicarbazide-sensitive amine oxidase (SSAO) plays a role in the in vivo and in vitro toxicity of several environmental and endogenous amines. We investigated the role of SSAO as a component of cell culture medium (through addition of fetal calf serum (FCS)) compared to intracellular SSAO in the in vitro cytotoxicity of three amines and metabolites. Smooth muscle cells and beating cardiac myocytes were grown in 96-well plates and exposed to various concentrations and combinations of FCS in medium, amines (allylamine, AA; benzylamine, BZA; and methylamine, MA), and amine metabolites (aldehydes: acrolein, benzaldehyde, and formaldehyde; hydrogen peroxide, H2O2; ammonia, NH3). Amine and amine metabolite cytotoxicity was quantified by monitoring cell viability. SSAO activity was measured in FCS, cardiovascular cells, or rat plasma by a radioenzymatic assay using [14C]BZA. Our data show that AA and its aldehyde metabolite, acrolein, were the most toxic compounds to both cell types. However, AA toxicity was FCS-dependent in both cell types, while BZA, MA, and amine metabolite (i.e., aldehydes, H2O2, and NH3) cytotoxicity showed little FCS dependence. In these experiments, medium containing 10% FCS had a calculated amine metabolic capacity that was 30- to 50-fold that of the cultured smooth muscle cellular content in a single well of a 96-well plate. Our study demonstrates that SSAO in FCS contributes to amine metabolism and cytotoxicity to rat cardiovascular cells in vitro and how critical it is to evaluate serum for its role in mechanisms of amine toxicity in vitro and in vivo.     

Mechanistic Studies on Nonviral Gene Delivery to the Intestine Using in Vitro Differentiated Cell Culture Models and an in Vivo Rat Intestinal Loop

Purpose. To identify factors influencing nonviral vector transfection in differentiated CaCo-2 and mucus-secreting coculture, CaCo-2:Ht29GlucH, cell culture models and to compare these in vitro results with in vivo transfection efficiency in rat intestine. Methods. A range of nonviral vectors including DOTAP, Lipofectin, Superfect, PEI, and polylysine were investigated. CaCo-2 and a mucus-secreting coculture were used at 21 days. Transfection efficiency was assessed using pCMVluc (firefly luciferase) plasmid, and radiolabeled plasmid was used to determine the binding and internalization of plasmid DNA. The in vivo model used was a ligated rat intestinal loop. Results. Transfection levels decreased by over 1000-fold in differentiated models relative to nondifferentiated COS-7 cells and were related to reductions in luciferase production by individual cells. Active internalization of DNA by the differentiated cells decreased. Removal of mucus by the mucolytic agent N-acetylcysteine, from the coculture system significantly reduced (p < 0.05) transfection efficiency. In vivo the transfection efficiency of PEI proved superior to DOTAP. Conclusions. Nonviral gene delivery to the hostile environment of the intestine is possible. Mechanistic studies using differentiated intestinal cell models aid identification of the rate-limiting steps to transfection and represent a more physiologically relevant approach to predict gene delivery to the intestine.     

ClC-3氯通道对H_2O_2诱导的大鼠主动脉平滑肌细胞凋亡的影响

目的研究C lC-3氯通道蛋白过表达对H2O2诱导的大鼠主动脉平滑肌细胞凋亡的影响。方法蛋白免疫印迹法检测C lC-3蛋白表达;形态学方法、DNA琼脂糖电泳、MTT法和流式细胞仪观察和分析H2O2诱导的大鼠主动脉平滑肌细胞形态学改变、DNA断裂、细胞存活率、凋亡率及C lC-3蛋白过表达对其影响。结果C lC-3蛋白过表达减轻H2O2诱导的大鼠主动脉平滑肌细胞形态学改变、DNA断裂及细胞凋亡率,增加细胞存活。结论提示C lC-3氯通道蛋白过表达保护H2O2诱导的大鼠主动脉平滑肌细胞凋亡。     

Chitosan enhances transfection efficiency of cationic polypeptides/DNA complexes

The aim of this research was to investigate the effect of cationic polypeptides mixed with chitosan (CS) on in vitro transfection efficiency and cytotoxicity in human cervical carcinoma cells (HeLa cells). The polypeptides/DNA complexes and ternary complexes (CS, polypeptides and DNA) at varying weight ratios were formulated and characterized by using gel electrophoresis. Their particle sizes and charge were evaluated. The effect of the type and molecular weight (MW) of polypeptides, the weight ratio, order of mixing, the pH and serum on transfection efficiency and cytotoxicity were evaluated in HeLa cells. Three types of polypeptides (poly-L-lysine; PLL, poly-L-arginine; PLA and poly-L-ornithine; PLO) were able to form complete complex with DNA at weight ratio above 0.1. The PLA MW >70 kDa showed the highest transfection efficiency. The order of mixing between CS, PLA and DNA affected the transfection efficiency. The highest transfection efficiency was observed in ternary complexes of PLA/DNA/CS (2:1:4) equal to PEI/DNA complex. For cytotoxicity studies, over 80% the average cell viabilities of the complexes were observed by MTT assay. This study suggests that the addition of CS to PLA/DNA is easy to prepare, safe and exhibits significantly improved DNA delivery potential in vitro.     

Establishment of evaluation method for siRNA delivery using stable cell line carrying the luciferase reporter gene

We determined the influence of siRNA (short interfering RNA) for expression of plasmid DNA (pDNA), when mismatched siRNA and pDNA encoding beta-galactosidase (beta-gal) were transfected into HeLa cells by the cotransfection method in which they were simultaneously added to the cells. Cationic liposomes (Lipofectamine2000) were used as a gene transfection reagent. The knockdown effect on beta-gal was observed even when mismatched siRNA was used, and the effect depended on the amount of added mismatched siRNA. But, there was not a distinct difference of introduction of pDNA into cells between using mismatched siRNA and without using it. We considered that the cotransfection method should be avoided when we confirm RNAi efficiency. The reliable evaluation method for siRNA delivery in vitro was thus established by using NFAT reporter HeLa stable cell line or CHO (pMAM-luc) cell line that had DNA encoding luciferase. The following experimental conditions for each cell line were optimized: cell numbers seeded, total incubation times, concentrations of added inducers, and incubation times after addition of inducers. Transfection performance was compared for six commercially available reagents by this method. No commercially available transfection reagent, however, could reduce luciferase activity by less than one tenth without causing cellular cytotoxicity. Development of novel reagents providing higher transfection effects without cytotoxicity is needed.     

CIC-3氯通道对H2O2诱导的大鼠主动脉平滑肌细胞凋亡的影响

目的研究CIC-3氯通道蛋白过表达对H2O2诱导的大鼠主动脉平滑肌细胞凋亡的影响。方法蛋白免疫印迹法检测CIC-3蛋白表达；形态学方法、DNA琼脂糖电泳、MTT法和流式细胞仪观察和分析H2O2诱导的大鼠主动脉平滑肌细胞形态学改变、DNA断裂、细胞存活率、凋亡率及CIC-3蛋白过表达对其影响。结果CIC-3蛋白过表达减轻H2O2诱导的大鼠主动脉平滑肌细胞形态学改变、DNA断裂及细胞凋亡率，增加细胞存活。结论提示CIC-3氯通道蛋白过表达保护H2O2诱导的大鼠主动脉平滑肌细胞凋亡。     

原花青素对人冠状动脉平滑肌细胞增殖和凋亡的影响

目的研究原花青素对人冠状动脉平滑肌细胞增殖和凋亡的影响。方法采用MTT实验检测原花青素对体外培养的人冠状动脉平滑肌细胞增殖的影响,运用流式细胞术、基因组DNA电泳观察凋亡特征性＂梯状＂条带检测细胞凋亡,采用Caspase活性定量检测试剂盒分析Caspase-9、Caspase-3的活性。结果原花青素对人冠状动脉平滑肌细胞具有明显的抑制作用,且其作用有剂量依赖性。流式细胞仪检测显示原花青素可以显著诱导人冠状动脉平滑肌细胞凋亡,DNA电泳显示原花青素作用于人冠状动脉平滑肌细胞后出现凋亡细胞特有的DNA阶梯状条带。原花青素作用后人冠状动脉平滑肌细胞Caspase-9、Caspase-3活性明显升高。结论原花青素可能通过线粒体信号通路抑制人冠状动脉平滑肌细胞的生长并诱导其凋亡。     

两种阳离子脂质体介导基因转染的比较研究

In order to study the important factors involved in cationic liposome-mediated gene transfer,   Lipofectamine 2000 or DOTAP was evaluated using three types of cells (Hep-2, MCF-7 and SW-480) in vitro transfection efficiencies.  Different properties of the two reagents were analyzed and compared by DNA     arrearage assay and MTT assay.  Both Lipofectamine 2000 and DOTAP had strong capability to combine with DNA; Lipofectamine 2000 can get higher transfection efficiency of the three cells by using GFP as report gene, meanwhile, DOTAP can also get higher transfection efficiency against Hep-2 cell.  However, DOTAP showed lower transfection efficiency against MCF-7 and SW-480 cell.  On the other hand, the cytotoxicity assay showed that over 85% cell viability of MCF-7 cell could be achieved both by Lipofectamine 2000 and DOTAP under the optimal transfection condition.  Relatively speaking, Lipofectamine 2000 has very high transfection efficiency in a broad range of cell lines, but because of the special selectivity of cell type on liposome, DOTAP also has a broad application prospect.     

Troglitazone induces apoptosis via the p53 and Gadd45 pathway in vascular smooth muscle cells

Thiazolidinediones, activators of peroxisome proliferator-activated receptor (PPAR)gamma, have been reported to induce apoptosis in many types of cells. In the present study, we investigated the effects of thiazolidinediones, troglitazone, and pioglitazone on the cell growth of vascular smooth muscle cells, and identified a specific effect of troglitazone in addition to PPARgamma activation. Subconfluent rat culture vascular smooth muscle cells were treated with or without PPARgamma activators, troglitazone (1-30 microM), or pioglitazone (1-30 microM) for 72 h. After treatment, cell viability was significantly reduced by troglitazone in concentrations of 5-30 microM but not by pioglitazone. Vascular smooth muscle cells appeared to float and shrink 48 h after treatment with 20 microM of troglitazone. In situ DNA labeling showed that the nuclei of these cells were positively stained, and genomic DNA extracted from the cells showed nucleosomal laddering. Messenger RNA expression levels of c-myc, p21, bax, bcl-2, and bcl-x were not changed by the treatment with troglitazone. In contrast, along with the induction of vascular smooth muscle cell apoptosis, both the mRNA and protein expression levels of p53 and Gadd45 markedly increased in response to troglitazone. These results strongly suggest that troglitazone can induce vascular smooth muscle cell apoptosis and that this effect is caused primarily by activation of the p53 and Gadd45 pathway but not by PPARgamma activation.     

Serotonin-induced proliferation of pulmonary arterysmooth muscle cells is serotonin transporter and ERK pathway dependent

AIM: To investigate the effect of serotonin transporter (5-HTT)inhibitor fluoxetine and antisense oligodeoxynucleotide (ODN)to extracelluar signal-regulated kinases (ERKs) on pulmonary arterial smooth muscle cells (PASMCs) proliferation induced by 5-HT. METHODS: Liposomal transfection was used to introduce ODNs to ERK1/2 into cultured rat PASMCs and the transfection efficiency was measured by observing the uptake of the     

Methylated N-(4-pyridinylmethyl) chitosan as a novel effective safe gene carrier

The objective of this study was to study the transfection efficiency of quaternized N-(4-pyridinylmethyl) chitosan; TM-Py-CS, using the pDNA encoding green fluorescent protein (pEGFP-C2) on human hepatoma cell lines (Huh 7 cells). The factors affecting the transfection efficiency, e.g. degree of quaternization (DQ), the extent of N-pyridinylmethyl substitution (ES) and weight ratio, have been investigated. The results revealed that TM-Py-CS was able to condense with pDNA. Illustrated by agarose gel electrophoresis, complete complexes of TM(69)Py(62)CS/DNA were formed at weight ratio above 1.1, whereas those of TM(53)Py(40)CS/DNA and TM(52)Py(13)CS/DNA were above 1.8 and 8, respectively. TM(69)Py(62)CS showed superior transfection efficiency to TM(53)Py(40)CS, TM(52)Py(13)CS, TM(65)CS and TM(43)CS at all weight ratios tested. The highest transfection efficiency of TM(69)Py(62)CS/DNA complexes was found at weight ratio of 4. The results indicated that the improved gene transfection was possibly due to 4-pyridinylmethyl substitution on CS which promoted the interaction and condensation with DNA as well as N-quaternization which increased CS water solubility. In cytotoxicity studies, high concentration of TM-Py-CS and TM-CS could decrease the Huh 7 cell viability. In conclusion, this novel CS derivative, TM(69)Py(62)CS, showed promising potential as a gene carrier by efficient DNA condensation and mediated higher level of gene transfection.     

Effect of calcitonin gene-related peptide on angiotensin II-induced proliferation of rat vascular smooth muscle cells

The present study was designed to examine the effect of calcitonin gene-related peptide (CGRP) on angiotensin II-induced proliferation of cultured rat vascular smooth muscle cells. Vascular smooth muscle cells were grown from explants of Sprague-Dawley rat aorta. Vascular smooth muscle cells (between passages 5 and 10) were incubated with 0.1% neonatal calf serum for 48 h, and then treated with angiotensin II (100 nM) in the absence or presence of CGRP for 24 h. The viability, DNA synthesis and cell cycle of vascular smooth muscle cells were measured. Western blotting was used to determine the activity of intracellular extracellular regulated kinase (ERK1/2). Angiotensin II significantly decreased the viability and proliferation of vascular smooth muscle cells, decreased the proliferation index, and increased the activity of ERK1/2; the effects of angiotensin II were inhibited by CGRP (1-100 nM) in a concentration-dependent manner. In conclusion, CGRP significantly inhibits angiotensin II-induced proliferation of vascular smooth muscle cells, an effect related to a decrease in the activation of mitogen-activated protein kinase pathway.     

Mannosylated polyethylenimine coupled mesoporous silica nanoparticles for receptor-mediated gene delivery

Organic-inorganic nanohybrids have been studied for their use as non-viral transfection agents. The purpose of this study was to examine the ability of mesoporous silica nanoparticles (MSN) coupled with mannosylated polyethylenimine (MP) to transfect plasmid DNA in vitro. Although MSN is biocompatible and has low cytotoxicity, it is not easily transfected into a variety of cell types. To overcome this barrier, MP was coupled to MSN (abbreviated as MPS) to target macrophage cells with mannose receptors and enhance transfection efficiency. The DNA conveyance ability of MPS was examined by evaluating properties such as particle size, zeta potential, complex formation, protection of plasmid DNA against DNase-I, and the release of DNA upon cell entry. Particle sizes of the MPS/DNA complexes decreased with increasing weight ratio of MPS to DNA, while the zeta potential increased. Complete MPS/DNA complexes were formed at a weight ratio of five, and their resistance to DNase-I was evaluated. Cytotoxicity studies showed that MPS/DNA complexes resulted in a high percentage of cell viability, compared with PEI 25K as a vector. The transfection efficiency of MPS/DNA complexes was evaluated on Raw 264.7 and HeLa cell lines. It was found that MPS/DNA complexes showed enhanced transfection efficiency through receptor-mediated endocytosis via mannose receptors. These results indicate that MPS can be employed in the future as a potential gene carrier to antigen presenting cells.     

Polyglutamic acid-based nanocomposites as efficient non-viral gene carriers in vitro and in vivo

A series of polyethylenimine (PEI) and γ-polyglutamic acid (PGA) nanocomposites (PPGA) was prepared and evaluated in terms of their cell viability and transfection efficiency in vitro and in vivo. On complexion with pDNA, the positively charged PPGA/DNA nanocomposites resulted in a higher level of in vitro reporter gene transfection (2.7-7.9-fold) as compared to native PEI, and selected commercial reagents and >95% cell viability in HEK293, HeLa and HepG2 cell lines. Further, PPGA-5 nanocomposite (the best working system in terms of transfection efficiency among the series) was found to efficiently transfect primary mouse keratinocytes up to 22% above the control level. PPGA-5, when tested for in vivo cytotoxicity in Drosophila, did not induce any stress in the exposed larvae in comparison with control. In vivo gene expression using PPGA-5 showed the highest transfection efficiency in spleen of mouse closely followed by heart tissues after intravenous injection through tail vein. Besides, these nanocomposites also delivered siRNA efficiently into mammalian cells, resulting in ∼80% suppression of EGFP expression. These results together demonstrated the potential of the projected nanocomposites for in vivo gene delivery.