Dynamic binding capacity of plasmid DNA in histidine-agarose chromatography

The use of histidine-agarose chromatography in the purification of supercoiled (sc) plasmid DNA (pDNA) from Escherichia coli lysates has been reported recently. In the current work we describe a set of breakthrough experiments which were designed to study the effect of parameters such as flow-rate, temperature, concentration and conformation on the dynamic binding capacity of pDNA to the histidine support. One of the most striking results shows that the dynamic binding capacity for sc pDNA decreases linearly from 250.8 to 192.0 microg sc pDNA/mL when the temperature is varied from 5 to 24 degrees C. This behaviour was attributed to temperature-induced, pre-denaturation conformational changes which promote the removal of negative superhelical turns in sc pDNA molecules and decrease the interaction of DNA bases with the histidine ligands. The capacity for sc pDNA was highly improved when using feeds with higher pDNA concentrations, a phenomenon which was attributed to the fact that pDNA molecules in more concentrated solutions are significantly compressed. A maximum capacity of 530.0 microg pDNA/mL gel was obtained when using a 125 microg/mL pDNA feed at 1 mL/min and 5 degrees C, a figure which is comparable to the plasmid capacity values published for other chromatographic supports. Finally, a more than 2-fold increase in capacity was obtained when changing from open circular to sc pDNA solutions. Overall, the results obtained provide valuable information for the future development and implementation of histidine chromatography in the process scale purification of pDNA.     

Comparison of different liquid chromatography stationary phases in LC-HRMS metabolomics for the detection of recombinant growth hormone doping control

Growth hormone (GH) is a polypeptide suspected of being used in horse racing to speed up physical performances. Despite scientific advances in the recent years, the control of its administration remains difficult. In order to improve it, a metabolomics study through LC-high resolution mass spectrometry measurements was recently initiated to assess the metabolic perturbations caused by recombinant equine growth hormone administration. Few tens of ions not identified structurally were highlighted as compounds responsible for the modification of metabolic profiling observed in treated animals. This previous work was based on the use of Uptisphere Strategy NEC as the chromatographic column. In parallel, more and more metabolomics studies showed the interest of the use of new chromatographic supports such as hydrophilic interaction chromatography for the analysis of polar compounds. It is in this context that an investigation was conducted on Uptisphere HDO and Luna hydrophilic interaction chromatography stationary phases to generate and process urinary metabolomics fingerprints, which could allow to establish a comparison with Uptisphere Strategy NEC. The chromatographic column the most adapted for the detection of new biomarkers of GH administration has been used to set up a relevant statistical model based on the analysis of more than hundred biological samples.     

Effective gene delivery into primary dendritic cells using synthesized PDMAEMA-iron oxide nanocubes

Dendritic cells (DCs) have been a target of vaccine delivery, gene therapy, and cancer immunotherapy. However, gene delivery to primary DCs using traditional non-viral molecules has been a difficult challenge. Herein we have developed a gene delivery system to primary DCs using magnetic iron oxide nanocubes (MCs) coated with cationic polymer under the induction of a magnetic field. The MCs were coated with positively charged polymer, poly(2-dimethylamino) ethyl methacrylate (MCs-PD) before the plasmid gene (pMAX-GFP) was adsorbed on their surfaces. Three different sizes (15, 40 and 90 nm) of MCs were synthesized, and subsequently, PDMAEMA was assembled onto the MC surfaces (MCs-PD). MCs-PD exhibited zeta potentials of +23 to +26 mV, and the obtained particles showed superparamagnetic character with saturation magnetization of 17–66 emu/g. The MCs-PD of 10–100 μg/mL showed low toxicity on bone marrow-derived dendritic cells (BMDCs) in MTT assay, and they were well taken up by BMDCs under a magnetic field. Moreover, the particles with small size exhibited the enhanced plasmid transfection efficiency without the activation of BMDCs. The MCs-PD could be a promising non-viral gene delivery system that helps to manipulate primary DCs in vitro, which will be beneficial for cell-based immunotherapy.