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Background: Intramuscular injection is a popular and effective approach to administer naked plasmid for transgene expression. The use of an adjuvant can provide a straightforward approach for enhancing transgene expression., Methods: Expression plasmid was formulated with various concentrations of trehalose for injection into the skeletal muscles of C57BL/6 mice. The effects of trehalose on gene dosage and the duration of transgene expression were assessed. The levels of transgene expression were indicated by levels of luciferase expression of the homogenized whole skeletal muscle or by histological X-gal staining of beta-galactosidase expression. Trehalose was also added to serum to examine the ability of protecting the DNA from degradation., Results: It was found that an optimal trehalose concentration of 10 mM will achieve a level of transgene expression that is seven-fold higher than in the absence of trehalose. When compared with other disaccharides, only the incorporation of trehalose can effectively enhance transgene expression. Trehalose is able to improve transgene expression by intramuscular injection at a low gene dosage as well as prolong the duration of transgene expression., Conclusions: Trehalose is an effective adjuvant for intramuscular administration of naked plasmid with respect to both enhanced levels and prolonged duration of transgene expression, most likely due to retarding plasmid degradation.

Using enhancers to improve the transfection efficiency of polyethylenimine (PEI) can circumvent the needs of chemical modifications as well as subsequent purification and characterization of the modified PEI. In this study, we found that incorporating trehalose into the transfection reagent could improve the transgene expression mediated by DNA-PEI complexes. Such enhancements were not observed when trehalose was replaced by other disaccharides. In an effort to explore the mechanisms, we examined how the timing of trehalose treatments and the durations of trehalose affected the percentages of cells expressing green fluorescent protein and the levels of intracellular ethidium monoazide labeled plasmid. Treatments with trehalose for 5-120 min prior to transfection could cause drops in transfection efficiency by 30-50%; such treatments, however, hardly affected the amounts of intracellular plasmid, indicating that the preexistence of intracellular trehalose could reduce transfection efficiency without lowering the endocytic activity. The transfection efficiency remained almost unchanged when the transfected cells were treated with trehalose after the removal of transfection reagents, indicating that trehalose had minimal effects on the machinery of protein synthesis. Despite the enhanced transgene expression, the presence of trehalose during transfection showed inhibitory effects on the internalization of DNA-PEI complexes. Additionally, the extent of enhancement in transgene expression strongly depended on the duration of trehalose. As the above observations suggested, only during the transfection process when complexes and trehalose coexisted, trehalose became an effective enhancer of transgene expression mediated by DNA-PEI complexes possibly by affecting the mechanisms of intracellular trafficking.

Branched polyethylenimine (PEI) is a cationic polymer capable of forming self-assembly complexes with DNA to become a highly efficient agent used in gene delivery. Conjugation through the primary amines of PEI is a most commonly used approach further to enable the targeting delivery or to improve the stability of the DNA-polymer complexes. An understanding of how the conjugation affects the transfection mechanisms can help in the design of efficient polycationic vectors. In order to investigate the effects of conjugation, folate and the dextrans of molecular weight 1500 (dex-1500) and 10 000 (dex-10000) were used to prepare three different types of PEI conjugates: dextran-PEI, folate-PEI, and folate-dextran-PEI, which were subsequently employed to form complexes with DNA. These conjugates were found to cause less cytotoxicity than the unmodified PEI as revealed by the MTT method, and to be able to deliver an approximate amount of ethidium monoazide labeled plasmid into the cells. The efficiencies of green fluorescent protein (GFP) expression mediated by these conjugates, however, were less efficient than those mediated by the unmodified PEI. A titration experiment suggested that conjugation through the primary amines of PEI resulted in the loss of relative buffering capacity, a major factor aiding the release of plasmid from the endosomes, presumably because the conjugated molecules hindered the protonation of the PEI conjugates. When a quantitative relationship between relative buffering capacity and transfection efficiency was examined, a threshold of relative buffering capacity, around 50% of the unmodified PEI, was noted to be required for minimal detection of GFP positive cells. In addition, the cytotoxicity could be also related to the relative buffering capacity in an approximately linear trend. It is thus concluded that the severe loss of relative buffering capacity by conjugation might be attributed to the inefficiency of transgene expression mediated by the dextran-PEI conjugates.

In vivo instability of a polycationic vector limits its efficacy after systemic administration. Conjugation of hydrophilic polymers with neutral charge onto polycationic vectors has been used to improve the stability by reducing the interactions between the vectors and the blood components, such as serum albumin. In this study, dextrans of molecular weight 10000 (dex-10000) and 1500 (dex-1500) were used to produce various degrees of grafting on linear and branched polyethylenimines (PEI), and the dextran-grafted polymers were used to prepare DNA-polymer complexes. The changes in size and in zeta-potential and the extent of DNA release after the exposure of the complexes to bovine serum albumin (BSA) were used to evaluate the stability of the complexes prepared at various ratios of DNA to polymer. Only the use of dextran-grafted branched PEI was found to be effective to improve the stability of the complexes in the presence of BSA. Dex-10000 was noted to provide a slightly better shielding than dex-1500 against the aggregation caused by BSA and helped maintain the sizes within 200 nm and the zeta-potentials close to neutral. It is thus concluded that the dextran-grafted branched PEI improved the stability of the DNA-polymer complexes and showed potential to conjugate with ligands for in vivo targeted gene delivery.

Ion-exchange chromatography is one of the most commonly used methods for plasmid preparation. In this study a modified method was used to purify plasmid from bacterial lysate using Q-Sepharose. Incorporation of alcohols into the washing buffers enhanced the separation of plasmid from RNA and proteins. The use of isopropanol and ethanol achieved a high yield and purity whereas the use of methanol failed to improve the plasmid purification using Q-Sepharose by batch adsorption-desorption. Stepwise elution containing various concentrations of isopropanol and NaCl was used in preparative chromatography to enhance the plasmid purification. The same stepwise elution was applied to the chromatography columns packed with 0.5, 20, and 200 ml of Q-Sepharose for plasmid purification from 7.5, 300, and 3000 ml bacterial broth, respectively. Complete separation of DNA from RNA and proteins was achieved under gravity flow by modulation of the alcohol concentrations in the stepwise elution. These three scales of chromatography maintained an approximate plasmid yield and the purified plasmid contained undetectable levels of RNA and protein.