A bench-scale protocol for the purification of plasmid DNA (pDNA) produced in Escherichia coli is described. The method is specifically designed to prepare pDNA vectors for gene therapy and DNA vaccination applications. The method comprises alkaline lysis, concentration with iso-propanol, prepurification by (NH4)2SO4 precipitation and purification by hydrophobic interaction chromatography (HIC), and desalting in gravity-operated 10-mL plastic columns. Analytical techniques used to control the performance of the method and to assess the quality of the pDNA vaccine are also described. Anion-exchange HPLC is used to determine pDNA concentration, whereas the presence of impurities such as RNA, proteins, E. coli genomic DNA, and endotoxins is determined by agarose gel electrophoresis, micro-bicinchoninic acid (BCA), real-time polymerase chain reaction and kinetic-quantitative chromogenic lymulus amoebacyte lysate (QCL LAL) assays, respectively. The method performs very well in terms of yield, purity and biological activity of the final pDNA. Furthermore, it is rapid, very easy to perform, and cost-efficient.
The efficacy of brain therapeutics is largely hampered by the presence of the blood-brain barrier (BBB), mainly due to the failure of most (bio) pharmaceuticals to cross it. Accordingly, this study aims to develop nanocarriers for targeted delivery of recombinant precursor microRNA (pre-miR-29b), foreseeing a decrease in the expression of the BACE1 protein, with potential implications in Alzheimer's disease (AD) treatment. Stearic acid (SA) and lactoferrin (Lf) were successfully exploited as brain-targeting ligands to modify cationic polymers (chitosan (CS) or polyethyleneimine (PEI)), and its BBB penetration behavior was evaluated. The intracellular uptake of the dual-targeting drug delivery systems by neuronal cell models, as well as the gene silencing efficiency of recombinant pre-miR-29b, was analyzed in vitro. Labeled pre-miR-29b-CS/PEI-SA-Lf systems showed very strong fluorescence in the cytoplasm and nucleus of RBE4 cells, being verified the delivery of pre-miR-29b to neuronal cells after 1 h transfection. The experiment of transport across the BBB showed that CS-SA-Lf delivered 65% of recombinant pre-miR-29b in a period of 4 h, a significantly higher transport ratio than the 42% found for PEI-SA-Lf in the same time frame. Overall, a novel procedure for the dual targeting of DDS is disclosed, opening new perspectives in nanomedicines delivery, whereby a novel drug delivery system harvests the merits and properties of the different immobilized ligands.
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