We report here on the analysis of synthetic oligonucleotides by electrospray ionization mass spectrometry (ESI-MS). After intensive removal of salt ions (especially sodium cations), negative ion mass spectra, allowing mass measurement with an accuracy of 0.01%, were obtained on several oligonucleotides up to 80 nucleotides. In most cases, the resolution was sufficient to observe n-1 and n-2 forms due to internal deletions during automated synthesis, and to identify the missing nucleotides. A 132-mer, whose size is close to the limit of automated chemical synthesis, was also successfully mass measured. A quantitative study showed that ESI-MS can provide quantitative data on oligonucleotides of similar size and structure. The described methodology is used to characterize oligonucleotide analogues such as phosphorothioate oligonucleotides designed for antisense applications. Finally, analyses in the positive ion mode on a trimer TpTpT in the presence of different amine bases were performed and allowed a better understanding of the influence of these bases on the ions formation.

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A novel type of synthetic vector, termed solvoplex, is described that can greatly enhance gene expression in lung after intrapulmonary delivery. Solvoplexes consist of plasmid DNA and organic solvents. Several organic solvents were analyzed, and luciferase reporter gene expression was observed after intrapulmonary delivery of solvoplexes containing DPSO (di-n-propylsulfoxide), TMU (tetramethylurea), or BMSO (butylmethylsulfoxide). Expression levels correlated with the amount of solvent used at constant DNA amounts. Highest expression was obtained in the lung after intratracheal injection with 15% DPSO resulting in an increase up to 440-fold compared with DNA alone. DPSO-solvoplexes (15%) gave higher reporter gene expression than polyplexes (ExGen 500) or lipoplexes (DOTAP-cholesterol or DOTAP-DOPE). Solvoplex-mediated gene expression did not depend on the delivery mode, and was observed in both mice and rats. Readministration of DPSO-solvoplexes was possible. A second injection after 4 weeks resulted in expression levels similar to the first administration. Histological analyses using lacZ and GFP reporter genes demonstrated gene expression in the lung airway epithelium after intratracheal and microspray delivery. When luciferase expression levels in lung homogenates were compared with adenovirus vectors, DPSO-solvoplexes were 4- or 100-fold less efficient, depending on the promoter used in the viral vector. A quantitative histological comparison between solvoplexes and adenovirus vectors in the best expressing regions revealed that solvoplexes yielded about 2% LacZ-positive cells in the lung airway epithelium, and adenovirus vectors about 20%. Using the microsprayer system, we demonstrated that DNA remained intact in solvoplexes on spraying and that reporter gene expression was observed in mice after intrapulmonary delivery of a solvoplex spray. DNA in DPSO-solvoplexes remained stable and functional after prolonged storage at room temperature.

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