Metal contaminants promote degradation of lipid/DNA complexes during lyophilization

Abstract

Oxidation reactions represent an important degradation pathway of nucleic acid-based pharmaceuticals. To evaluate the role of metal contamination and chelating agents in the formation of reactive oxygen species (ROS) during lyophilization, ROS generation and the stability of lipid/DNA complexes were investigated. Trehalose-containing formulations were lyophilized with different levels of transition metals. ROS generation was examined by adding proxyl fluorescamine to the formulations prior to freeze-drying. Results show that ROS were generated during lyophilization, and both supercoil content and transfection rates decreased as the levels of metal-induced ROS increased. The experiments incorporating chelators demonstrated that some of these agents (e.g., DTPA, desferal) clearly suppress ROS generation, while others (e.g., EDTA) enhance ROS. Surprisingly, there was not a strong correlation of ROS generated in the presence of chelators with the maintenance of supercoil content. In this study, we demonstrated the adverse effects of the presence of metals (especially Fe(2+)) in nonviral vector formulations. While some chelators attenuate ROS generation and preserve DNA integrity, the effects of these additives on vector stability during lyophilization are difficult to predict. Further study is needed to develop potent formulation strategies that inhibit ROS generation and DNA degradation during lyophilization and storage.

Fe³⁺-dependent epistasis between the CpxR-activated loci and the PmrA-activated LPS modification loci in <i>Salmonella enterica</i>

“…In addition, it may suggest an alternative mode of Fe 3+ entry into the periplasm in which the Fe 3+ /pyrophosphate complex tears off slowly under an aerobic condition in the presence of a reducing power from an unknown resource at a physiological temperature. Note that chelated iron in the presence of oxygen and a reducing agent can lead to repetitive cleavage of macromolecules such as DNA (Enright et al, 1992;Molina and Anchordoquy, 2007) and that an Fe 3+ /ADP chelate complex can cause lipid peroxidation of a mitochondrial lipid in a reducing agent-dependent manner in Eucaryotes (Takeshige et al, 1980). However, it is also true that Fe 3+ -mediated bacterial cell killing of the pmrA mutant can still occur even under anaerobic conditions in a growth-dependent manner (Chamnongpol et al, 2002).…”

Stabilization of Plasmid DNA and Lipid-Based Therapeutics as Dehydrated Formulations

“…However, DNA must resist the freezing and drying stresses encountered during processing before stability can be addressed. In this respect, it has been demonstrated that the addition of disaccharides, such as sucrose and trehalose, are beneficial to prevent loss of SC DNA during lyophilization [55,56,82,91,96,113,115,121,178,179].…”

“…Considering that biomolecules are immobilized in a glassy-excipient phase where reactions requiring positional specificity would be considerably inhibited in the dried state [127], our recent findings show that degradation can still occur at significant rates despite maintenance of a glassy phase (e.g., in trehalose). In fact, our validated fluorescent-based method [115] has been used consistently to detect the formation of ROS in lyophilized naked DNA, lipid alone, as well as lipid/DNA complexes, and demonstrated that ROS are still active in the solid state [115,178,179,183]. Furthermore, our findings suggest that trace metal contamination (Cu ) is a prominent factor affecting the stability of these biomolecules, particularly when an unsaturated lipid is present in the formulation composition (e.g., dried lipid/DNA complexes, as described in corresponding section of this chapter) [179,183].…”

“…Furthermore, our findings suggest that trace metal contamination (Cu ) is a prominent factor affecting the stability of these biomolecules, particularly when an unsaturated lipid is present in the formulation composition (e.g., dried lipid/DNA complexes, as described in corresponding section of this chapter) [179,183]. It should also be noted that even very small quantities (parts per billion, ppb) of transition metals (e.g., ferrous iron −Fe 2+ ) can mediate generation of ROS in the dried state [115]. Furthermore, trace amounts of metal contaminants are present even in the cleanest materials available commercially (e.g., clinical-grade DNA and GMP-grade sugars) [103,115,183].…”

“…Despite the growing body of research on nonviral vector dehydration, to date, lyophilization studies have mostly focused on the stability of vectors (e.g., naked DNA [91,[110][111][112][113][114], lipoplexes [83,86,[115][116][117], polyplexes [85,88,99,109,118,119], and lipopolyplexes [95,120]) during acute freeze-drying stress. Considering the complexity involved in the stabilization of individual vectors (e.g., naked DNA, lipid) as dehydrated formulations, this chapter addresses these challenges separately.…”