Stabilization of DNA utilizing divalent cations and alcohol

• Published in: International journal of pharmaceutics Vol. 264; no. 1; pp. 15 - 24
• Main Authors: Knight, Jefferson D, Adami, Roger C
• Format: Journal Article
• Language: English
• Published: Amsterdam Elsevier B.V 02.10.2003; Elsevier
• Subjects: Animals; Biological and medical sciences; Cations, Divalent - chemistry; Cats; Chemistry, Pharmaceutical; DNA; DNA - chemistry; Drug Stability; Erythropoietin - genetics; Gene therapy; General pharmacology; Ion-pair complex; Kinetics; Medical sciences; Microscopy, Electron; Particle Size; Pharmacology. Drug treatments; Physicochemical properties. Structure-activity relationships; Solvent; Solvents - chemistry; Spectrometry, Fluorescence; Stabilization; Stress, Mechanical; tert-Butyl Alcohol - chemistry; Ion-pair complex; Gene therapy; Stabilization; Solvent; DNA; Self assembly; Stability; Pharmaceutical technology; Ion pair; Prevention; Butanol derivatives; Shear stress; Calcium Chlorine; Divalent cation; Surface potential; Physicochemical properties
• ISSN: 0378-5173; 1873-3476
• DOI: 10.1016/S0378-5173(03)00367-3

A novel method for protection of DNA from high shear induced damage is presented. This method uses simple divalent cations and the lyophilizable alcohol, tert-butanol, to self-assemble DNA into condensed, shear-resistant forms. The DNA used in these studies was a 5600 BP plasmid DNA encoding a therapeutic gene. Various solvents and salts were used to identify optimal conditions to condense plasmid DNA. A stable formulation was identified with plasmid DNA condensed in a cosolvent solution containing 20% (v/v) tert-butanol and 1 mM calcium chloride. The DNA was formulated at 100 μg/ml and condensed into rod and toroidal shapes that were approximately 50–300 nm in diameter. The rods were found to be kinetically stable for greater than 24 h following their preparation. Condensation of the plasmid DNA in this manner results in nearly 100% of the plasmid DNA remaining intact after 1 min of high shear stress applied by a 50 W probe sonicator. Uncondensed control plasmid DNA is completely fragmented following 30 s of identical sonication. It is believed that condensation of DNA in this manner will permit utilization of high shear-stress inducing processing techniques, such as lyophilization or spray-drying without resulting in damage to the DNA.