The impact of fluid-dynamic-generated stresses on chDNA and pDNA stability during alkaline cell lysis for gene therapy products.

Extensive tests have been carried out to assess the impact of fluid-dynamic-generated stress during alkaline lysis of Escherichia coli cells (host strain DH1 containing the plasmid pTX 0161) to produce a plasmid DNA (pDNA) solution for gene therapy. Both a concentric cylinder rheometer and two stirred reactors have been used, and both the alkaline addition and neutralization stages of lysis have been studied. Using a range of shear rates in the rheometer, stirrer speeds in the reactors, and different periods of exposure, their impact on chromosomal DNA (chDNA) and pDNA was assessed using agarose gel electrophoresis, a Qiagen Maxiprep with a polymerase chain reaction (PCR) assay, and a Qiagen Miniprep purification with a UV spectrophotometer. Comparison has been made with unstressed material subjected to similar holding times. These tests essentially show that under all these conditions, <2% chDNA was present in the pDNA solution, the pDNA itself was not fragmented, and a yield of 1 mg/g cell was obtained. These results, together with studies of rheological properties, have led to the design of a 60-L, stirred lysis reactor and the production of high-quality pDNA solution with <1% chDNA after further purification.     

Formulation and in vitro transfection efficiency of poly (D, L-lactideco-glycolide) microspheres containing plasmid DNA for gene delivery

The stability, in vitro release, and in vitro cell transfection efficiency of plasmid DNA (pDNA) poly (D,L.-lactide-co-glycolide) (PLGA) microsphere formulations were investigated. PLGA microspheres containing free and polylysine (PLL)-complexed pDNA were prepared by a water-oil-water solvent extraction/evaporation technique. Encapsulation enhanced the retention of the supereoiled structure of pDNA as determined by gel electrophoresis. PLL complexation of pDNA prior to encapsulation increased both the stability of the supercoiled form and the encapsulation efficiency. Free pDNA was completely degraded after exposure to DNase while encapsulation protected the pDNA from enzymatic degradation. Rapid initial in vitro release of pDNA was obtained from microspheres containing free pDNA. while the release from microspheres containing PLL-complexed pDNA was sustained for more than 42 days. Bioactivity of encapsulated pDNA determined by in vitro cell transfection using Chinese hamster ovary cells (CHO) showed that the bioactivity of encapsulated pDNA was retained in both formulations but to a greater extent with PLL-complexed pDNA microspheres. These results demonstrated that PLGA microspheres could be used to formulate a controlledrelease delivery system for pDNA that can protect the pDNA from DNase degradation without loss of functional activity.     

Copper(II) complexes of diclofenac: Spectroscopic studies and DNA strand breakage

Copper(II) complexes of diclofenac with interesting anti-inflammatory profiles have been prepared and studied by infrared and electronic spectroscopy. In the solid state and in polar and coordinating solvents, all the complexes are solvated binuclear carboxylato-bridged complexes, [Cu(L)²(S)]², where L is monodeprotonated diclofenac and S is the axially bonded solvent. The effect of the copper(II) complexes on the in vitro DNA strand breakeage was studied by agarose gel electrophoresis. Relaxation or double stranded scissions of pDNA were observed leading to the formation of linear pDNA. Treatment of pDNA with high concentrations of these compounds caused a disappearance of pDNA. For the parent drug, sodium diclofenac, no effect on the pDNA was observed. This study presents some indications that the binuclear copper(II) complexes, [Cu(L)²(S)]², could have some relevance in the treatment of tumor cell lines.     

机械喷射式雾化机对质粒DNA完整性的影响及保护性研究

目的:探索目前临床广泛使用的喷射式雾化机对质粒DNA(pDNA)完整性破坏的影响及保护措施。方法:在研究雾化时间对pDNA完整性影响的实验中,加入5ml pDNA(20μg/ml),分别在开始雾化后收集第1min内;第2min内;第3min内;第4min内;第5min内及第10min内雾化器喷口处的雾化液滴;在研究加样量对裸pDNA完整性影响的实验中,分别取2ml、4ml、6ml、8ml,各雾化4min,收集最后一分钟内雾化液滴。在两种高分子聚合物对pDNA保护性研究的实验中,各取4ml未经高分子聚合物修饰的以及经过聚乙烯亚胺(polyethylenimine, PEI)或阳离子脂质体修饰后的pDNA,分别雾化10min,收集最后一分钟内雾化液滴。使用琼脂糖凝胶电泳分析雾化样本质粒完整性。结果:在雾化时间由1min增至10min后完整性部分所占百分比由(83.5±2.2)%降至(37.1±2.8)%;加样量由2ml 增至8ml 后,pDNA完整性部分所占百分比由(32.1±3.5)%增至(93.6±0.6)%;经过PEI或阳离子脂质体修饰后的pDNA在雾化过程中几乎无破坏现象。结论:喷射式雾化机对pDNA的破坏呈剂量、时间依赖性,PEI与阳离子脂质体对pDNA保护效果良好,为喷射式雾化机在基因雾化治疗中的应用打下一定基础。     

气道内微量雾化机对质粒DNA完整性和基因转染效率的影响

目的:探索新型气道内微量雾化机对质粒DNA(pDNA)完整性和整体动物基因转染效率的影响。方法:首先,研究新型气道内微量雾化机对pDNA破坏程度。分别向新型气道内微量雾化机和目前临床普遍使用的机械喷射式雾化机的加药池内加入2ml pDNA(20μg/ml),在开始雾化后第1min、3min、5min分别收集两种雾化机嘴处的雾化液滴,用琼脂糖凝胶电泳观察比较质粒的完整性。其次,研究整新型气道内微量雾化机在整体动物上对基因转染效率的影响。分别用新型气道内微量雾化机和目前临床普遍使用的机械喷射式雾化机,给大鼠雾化经多聚乙烯亚胺(polyethylenimine,PEI)修饰的相同量的绿色荧光蛋白质粒(plasmid DNA of green fluorescent protein gene, pEGFP)3.3μg(PEI/pEGFP),24h后提取动物肺组织进行反转录,用real time PCR及琼脂糖凝胶电泳观察分析绿色荧光蛋白(green fluorescent protein, GFP)的mRNA表达情况。结果:新型气道内微量雾化机在雾化第1min、第3min、第5min后完整的质粒比例分别为(99.6±0.7)%、(100±1.2)%、(99.6±0.7)%,与未雾化对照相比(P>0.05,n=3),无统计学差异,新型雾化机对质粒破坏性可以忽略。临床常用的喷射式雾化机在相同时间段内完整的质粒比例分别为(70.3±1.5)%、(49.3±1.5)%、(32.7±0.6)%。与未雾化对照相比(P<0.05,n=3)有统计学差异,并且随雾化时间增加临床常用的喷射式雾化机对质粒的完整性破坏程度逐渐增加;新型气道内微量雾化机的基因转染效率高于临床普遍使用的机械喷射式雾化机转染效率的(382.1±101.1)倍(P<0.01,n=3)。结论:新型气道内微量雾化机对质粒没有破坏性,能显著增加雾化吸入基因转染效率。为雾化基因治疗提供了一种合适的工具。     

Magnetic nano-Fe3O4 particles targeted gathering and bio-effects on nude mice loading human hepatoma Bel-7402 cell lines model under external magnetic field exposure in vivo

Abstract

Magnetic nano-Fe₃O₄ particles (MNPs), static magnetic field (SMF) and extremely low-frequency altering electric magnetic field (ELFF) were utilized to treat nude mice loading hepatoma Bel-7402 cell lines to investigate the therapeutic values of MNPs combined with ELFF in vivo. Magnetic resonance image (MRI) figures showed that about 98.9% MNPs injected into mice body through tail vein were gathered in tumor focal by SMF directing exposure. Single ELFF and MNPs treatments did not influence mice physiological function obviously. However, gathered MNPs combined with ELFF treatment prolonged mice survival time and inhibited loading tumor cells proliferation significantly compared to other mice groups (p?<?0.05); furthermore, the tumor cells early apoptosis ratio of mice group was significantly higher than other groups (p?<?0.05), and ELFF combined with gathered MNPs treatment improved tumor cells early apoptosis associated with Bcl group protein expression: Bax protein expression was higher than Bcl-2 and the combined treatment improved cells Heat shock protein-27 (Hsp-27) expression which could protect cells avoiding early apoptosis. The possible mechanism that this kind of combination inducing more cells into early apoptosis could be due to ELFF exposure influencing cells ion metabolism, MNPs strengthening the effects, and the ELFF vibrating MNPs to generate extra heat and activate cellular heat shock signal channel.     

Molecular recognition of oligonucleotides and plasmid DNA by l‐methionine

The present study explores the effect of oligonucleotide composition on the mechanism of retention to l ‐methionine agarose support by chromatography and saturation transfer difference (STD)‐nuclear magnetic resonance (NMR) techniques. All chromatographic experiments were performed using 1.5 M (NH₄)₂SO₄. The binding profiles obtained by chromatography show that oligonucleotides with thymine had the highest retention time. In general, the larger homo‐oligonucleotides are more retained to the l ‐methionine agarose support. Moreover, the study with hetero‐oligonucleotides confirms that the presence of guanine reduces the retention on the l ‐methionine chromatographic support. These results are in accord with STD‐NMR experiments, which show that the strongest signals were observed for the methyl group of thymine, and no STD signals were observed for the guanosine protons. Finally, the retention behaviour of linear plasmid DNA (pDNA) with different sizes and base composition (2.7‐kbp pUC19, 6.05‐kbp pVAX1‐LacZ, 7.4‐kbp pVAX1‐LacZgag and 14‐kbp pcDNA‐based plasmid) was also evaluated by chromatography. The results indicate that the underlying mechanism of retention involves not only hydrophobic interactions but also other elementary interactions responsible for the biorecognition of pDNA molecules by l ‐methionine ligands. Copyright © 2014 John Wiley & Sons, Ltd.     

Separation of supercoiled from open circular forms of plasmid DNA,and biological activity detection

To establish a cost-effective purification process for the large-scale production of plasmid DNA for gene therapy and DNA vaccination, a single anion-exchange chromatography (AEC) step was employed to purify supercoiled plasmid DNA (sc pDNA) from other isoforms and Escherichia coli impurities present in a clarified lysate. Two different size and conformation plasmids were used as model targets, and showed similar elution behavior in this chromatographic operation, in which sc pDNA was effectively separated from open circle plasmid DNA (oc pDNA) in a salt gradient. The process delivered high-purity pDNA of homogeneity of 95 ± 1.1% and almost undetectable levels of endotoxins, genomic DNA, RNA and protein, at a yield of 65 ± 8%. Furthermore, the transfection efficiency (29 ± 0.4%) was significantly higher than that (20 ± 0.1%) of a pDNA control. The present study confirms the possibility of using a single AEC step to purify sc pDNA from other isoforms and host contaminants present in a clarified E. coli lysate.     

Microparticle‐mediated gene delivery for the enhanced expression of a 19‐kDa fragment of merozoite surface protein 1 of Plasmodium falciparum

The 19 kDa carboxyl‐terminal fragment of merozoite surface protein 1 (MSP1₁₉) is a major component of the invasion‐inhibitory response in individual immunity to malaria. A novel ultrasonic atomization approach for the formulation of biodegradable poly(lactic‐co‐glycolic acid) (PLGA) microparticles of malaria DNA vaccines encoding MSP1₁₉ is presented here. After condensing the plasmid DNA (pDNA) molecules with a cationic polymer polyethylenimine (PEI), a 40 kHz ultrasonic atomization frequency was used to formulate PLGA microparticles at a flow rate of 18 mL h^?1. High levels of gene expression and moderate cytotoxicity in COS‐7 cells were achieved with the condensed pDNA at a nitrogen to phosphate (N/P) ratio of 20, thus demonstrating enhanced cellular uptake and expression of the transgene. The ability of the microparticles to convey pDNA was examined by characterizing the formulated microparticles. The microparticles displayed Z‐average hydrodynamic diameters of 1.50–2.10 μm and zeta potentials of 17.8–23.2 mV. The encapsulation efficiencies were between 78 and 83%, and 76 and 85% of the embedded malaria pDNA molecules were released under physiological conditions in vitro. These results indicate that PLGA‐mediated microparticles can be employed as potential gene delivery systems to antigen‐presenting cells in the prevention of malaria. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2010     

Determination of the effective diffusion coefficient of oxygen in gel materials in relation to gel concentration

Summary The effective diffusion coefficient of oxygen, ID_e, was determined in different gel support materials (calcium alginate, -carrageenan, gellan gum, agar and agarose) which are generally used for immobilization of cells. The method used was based upon fitting Crank's model on the experimental data. The model describes the solute diffusion from a well-stirred solution into gel beads which are initially free of solute. The effect of the gel concentration on ID_e of oxygen in the gel was investigated. The results showed a decreasing ID_e for both agar and agarose at increasing gel concentration. In case of calcium alginate and gellan gum, a maximum in ID_e at the intermediate gel concentration was observed. It is hypothesized that this phenomenon is due to a changing gelpore structure at increasing gel concentrations. The ID_e of oxygen in calcium alginate, -carrageenan and gellan gum varied from 1.5^*10^–9 to 2.1^*10^–9 m²s^–1 in the gel concentration range of 0.5 to 5% (w/v).     

Effect of experimental treatment on GAPDH mRNA expression as a housekeeping gene in human diploid fibroblasts

Background  

Several genes have been used as housekeeping genes and choosing an appropriate reference gene is important for accurate quantitative RNA expression in real time RT-PCR technique. The expression levels of reference genes should remain constant between the cells of different tissues and under different experimental conditions. The purpose of this study was to determine the effect of different experimental treatments on the expression of glyceraldehyde 3-phosphate dehydrogenase (GAPDH) mRNA so that the reliability of GAPDH as reference gene for quantitative real time RT-PCR in human diploid fibroblasts (HDFs) can be validated. HDFs in 4 different treatment groups viz; young (passage 4), senescent (passage 30), H₂O₂-induced oxidative stress and γ-tocotrienol (GTT)-treated groups were harvested for total RNA extraction. Total RNA concentration and purity were determined prior to GAPDH mRNA quantification. Standard curve of GAPDH expression in serial diluted total RNA, melting curve analysis and agarose gel electrophoresis were used to determine the reliability of GAPDH as reference gene.     

Superporous agarose monoliths as mini-reactors in flow injection systems

A new type of agarose material, superporous agarose, was used as a support material in an analytical system designed for monitoring of bioprocesses with respect to metabolites and intracellular enzymes. The superporous agarose was used in the form of miniaturised gel plug columns (15×5.0 mM I.D. monolithic gel bed). The gel plugs were designed to have one set of very large pores (about 50 m in diameter) through which cells, cell debris and other particulate contaminants from the bioreactor could easily pass. The material also had normal diffusion pores (300 Å) characteristic of all agarose materials, providing ample surface for covalent attachment of antibodies and enzymes used in the analytical sequence. The superporous agarose gel plug columns were characterised with respect to flow properties and handling of heavy cell loads as well as dispersion of injected samples (a Bodenstein number of about 40 was observed with acetone tracer at a flow rate of 1 ml min^–1). To evaluate the practical performance of the superporous gel plug columns, two applications were studied: (1) on-line determination of glucose in cultivation broth (gel plug with immobilized glucose oxidase) and (2) immunochemical quantification of intracellular -galactosidase in E. coli (gel plug with lysozyme to achieve cell lysis and gel plug with antibodies against -galactosidase).     

An improved purification and further characterization of the messenger ribonucleic acid for the fatty acid synthetase from rat liver

High purity fatty acid synthetase mRNA has been prepared from rat liver. The translational purity of the mRNA preparation was at least 27% as judged by the percentage of the radioactivity incorporated into acid-insoluble material that was precipitated by anti-fatty acid synthetase antibody. The specific activity of the mRNA was 220-times greater than that reported previously from this laboratory [1]. The large increase in the specific activity was achieved by the repeated use of high resolution linear-log sucrose density gradient centrifugation and the removal of 28 S rRNA by Sepharose 4B chromatography, as well as by the optimization of the K⁺ concentration (160 mM) in the reticulocyte lysate translation system. The mRNA preparation showed a single major band on agarose gel electrophoresis under denaturing conditions, and the translational activity of the fatty acid synthetase mRNA on the gel was found to coincide with this band. The molecular weight of the fatty acid synthetase mRNA is 2.5·10⁶ Da. The mRNA directed the synthesis of fatty acid synthetase with a molecular weight indistinguishable from that of the authentic enzyme subunit (M_r = 240 000). The copurification of the translation product and authentic enzyme revealed that the fatty acid synthetase polypeptides synthesized in the reticulocyte lysate system are assembled in vitro into dimers, the native form of the enzyme.     

On the stability of plasmid DNA vectors during cell culture and purification

Gene therapy and DNA vaccination applications have increased the demand for highly purified plasmid DNA (pDNA) in the last years. One of the main problems related to the scale-up of pDNA purification is the degradation of the supercoiled (sc) isoforms during cell culture and multi-stage purification. In this work, a systematic study of the stability of two model plasmids (3,697 and 6,050 bp) during a mid-scale production process, which includes fermentation, alkaline lysis, isopropanol and ammonium sulphate precipitation and hydrophobic interaction chromatography, was performed. Results indicate that by extending cell culture (up to 26 h) and cell lysis (up to 2 h) it is possible to significantly reduce the amounts of RNA, without significantly compromising the yields of the sc pDNA isoform, a feature that could be conveniently exploited for downstream processing purposes. The stability of pDNA upon storage of E. coli pellets at different temperatures indicates that, differently from RNA, pDNA is remarkably stable when stored in cell pellets (>3 weeks at 4°C, >12 weeks at −20°C) prior to processing. With alkaline lysates, however, storage at −20°C is mandatory to avoid sc pDNA degradation within the first 8 weeks. Furthermore, the subsequent purification steps could be carried out at room temperature without significant pDNA degradation. Since the unit operations and process conditions studied in this work are similar to those generally used for plasmid DNA production, the results presented here may contribute to improve the current knowledge on plasmid stability and process optimization. Authors Freitas and Azzoni contributed equally to this work.     

Bacteria capture, lysate clearance, and plasmid DNA extraction using pH-sensitive multifunctional magnetic nanoparticles

A multifunctional magnetic nanoparticle (MNP)-assisted bioseparation method was developed to isolate plasmid DNA (pDNA) from Escherichia coli culture. Using the pH-sensitive carboxyl-modified magnetic nanoparticles, both cell capture and the subsequent removal of genomic DNA/protein complex after lysis can be achieved simply by magnetic separation. Furthermore, the yield and purity of pDNA extracted by MNPs are comparable to those obtained using organic solvents or commercial kits. This time- and cost-effective protocol does not require centrifugation or precipitation steps and has the potential for automated DNA extraction, especially within miniaturized lab chip applications.     

Effect of Coccolith Polysaccharides Isolated from the Coccolithophorid, <Emphasis Type="Italic">Emiliania huxleyi</Emphasis>, on Calcite Crystal Formation in In Vitro CaCO<Subscript>3</Subscript> Crystallization

Marine coccolithophorids (Haptophyceae) produce calcified scales “coccoliths” which are composed of CaCO₃ and coccolith polysaccharides (CP) in the coccolith vesicles. CP was previously reported to be composed of uronic acids and sulfated residues, etc. attached to the polymannose main chain. Although anionic polymers are generally known to play key roles in biomineralization process, there is no experimental data how CP contributes to calcite crystal formation in the coccolithophorids. CP used was isolated from the most abundant coccolithophorid, Emiliania huxleyi. CaCO₃ crystallization experiment was performed on agar template layered onto a plastic plate that was dipped in the CaCO₃ crystallization solution. The typical rhombohedral calcite crystals were formed in the absence of CP. CaCO₃ crystals formed on the naked plastic plate were obviously changed to stick-like shapes when CP was present in the solution. EBSD analysis proved that the crystal is calcite of which c-axis was elongated. CP in the solution stimulated the formation of tabular crystals with flat edge in the agarose gel. SEM and FIB-TEM observations showed that the calcite crystals were formed in the gel. The formation of crystals without flat edge was stimulated when CP was preliminarily added in the gel. These observations suggest that CP has two functions: namely, one is to elongate the calcite crystal along c-axis and another is to induce tabular calcite crystal formation in the agarose gel. Thus, CP may function for the formation of highly elaborate species-specific structures of coccoliths in coccolithophorids.     

巯基化壳聚糖季铵盐用于基因输送的研究

目的:本研究诣在对壳聚糖进行修饰,以解决其水溶性问题和基因释放困难的问题。方法:本研究通过2,3-环氧丙基三甲基氯化铵和N-乙酰-L-半胱氨酸对壳聚糖进行修饰,得到巯基化壳聚糖季铵盐(TMC-SH),使其在生理条件下带正电并含有一定量的游离巯基。以TMC-SH为基因载体,形成基因复合物。通过琼脂糖凝胶电泳考察其稳定性,并测定其粒径和ζ-电位。通过DTT条件下的粒径测定,考察基因复合物的还原响应性。结果:核磁结果表明合成TMC-SH的季铵盐取代度为22%,游离巯基-SH含量为79.22μmol/g;琼脂糖凝胶电泳结果表明以TMC-SH为载体形成的二硫键交联的基因复合物TMC-SS/p DNA具有较好的稳定性;而且,二硫键交联以后基因复合物粒径较小,结构更为密实;在还原条件下粒径变大,表明二硫键交联的基因复合物变得疏松,说明其粒径具有还原响应性。结论:对壳聚糖进行低取代度的季铵盐修饰和一定量的巯基化修饰后,其具有较好的包载p DNA能力和还原响应性的基因释放能力。     

Determination of Acidic Component of Konjac Mannan

mRNA was isolated from mammary glands of lactating cow by affinity chromatography on poly(U)-Sepharose. The mRNA was heterogeneous on 3% agarose gel electrophoresis in the presence of 6m urea. The molecular weight of the main peak was estimated to be 3.3 x 10⁵. The mRNA was translated in a cell-free protein synthesizing system derived from wheat germ extract, and the translation products were analysed by the indirect immunoprecipitation method using specific antisera for casein components. About 50% of the total protein directed by this mRNA was casein. The relative amounts of α_s1-, β-,and k-casein in the translation products were nearly the same as those in bovine milk. The immunoprecipitates were analysed on sodium dodecyl sulfatepolyacrylamide gradient gel (15~20%) electrophoresis, and their mobilities were compared with those of dephosphorylated and non-glycosylated casein as standard, α_s1- and k Casein synthesized in vitro migrated more slowly than standard caseins, while synthesized β-casein migrated slightly faster than the standard β-casein.