How high energy fluxes may affect Rayleigh-Taylor instability growth in young supernova remnants.

Energy-transport effects can alter the structure that develops as a supernova evolves into a supernova remnant. The Rayleigh-Taylor instability is thought to produce structure at the interface between the stellar ejecta and the circumstellar matter, based on simple models and hydrodynamic simulations. Here we report experimental results from the National Ignition Facility to explore how large energy fluxes, which are present in supernovae, affect this structure. We observed a reduction in Rayleigh-Taylor growth. In analyzing the comparison with supernova SN1993J, a Type II supernova, we found that the energy fluxes produced by heat conduction appear to be larger than the radiative energy fluxes, and large enough to have dramatic consequences. No reported astrophysical simulations have included radiation and heat conduction self-consistently in modeling supernova remnants and these dynamics should be noted in the understanding of young supernova remnants.

Degradation of lyophilized lipid/DNA complexes during storage: the role of lipid and reactive oxygen species.

The presence of trace amounts of metal ions in nonviral vector formulations can significantly affect the stability of lipid/DNA complexes (lipoplexes) during acute freeze-drying. The goal of the present study was to evaluate the generation of reactive oxygen species (ROS) in dried formulations of lipoplexes and in their individual components (lipid or naked DNA). The experiments were conducted in the presence or absence of a transition metal (Fe2+). Lipoplexes and their individual components were formulated in trehalose and subjected to lyophilization and stored for a period of up to 2 months at +60 degrees C. Physico-chemical characteristics and biological activity were evaluated at different time intervals. Generation of ROS during storage was determined by adding a fluorescence probe to the formulations prior to freeze-drying. We also monitored the formation of thiobarbituric reactive substances (TBARS). Our results show that ROS and TBARS form during storage in the dried state. Our findings also suggest that degradation is more rapid in the presence of lipid, even in the absence of metal. We also showed that dried naked DNA formulations are more stable without the lipid component. Effective strategies are then needed to minimize the formation and accumulation of oxidative damage of lipoplexes during storage.

Formulation strategies to minimize oxidative damage in lyophilized lipid/DNA complexes during storage.

It has been shown that degradation of lipid/DNA complexes (lipoplexes) continues in the dried state during storage. The goal of this study was to evaluate the ability of various strategies to minimize the formation of reactive oxygen species (ROS) in lyophilized lipoplexes during storage, including metal removal from reagents, air displacement, and fortification with chelator/antioxidant agents. Formulations containing individual chelator (DTPA) and antioxidants (L-methionine or alpha-tocopherol), or in combination, were subjected to lyophilization. Accelerated storage conditions were investigated and physico-chemical characteristics and biological activity of samples were monitored at different time intervals. Generation of ROS during storage was determined by adding proxyl fluorescamine to the formulations prior to freeze-drying. Lipid peroxidation was assessed by monitoring the formation of thiobarbituric reactive substances (TBARS) and lipid hydroperoxides. We also assessed the effect of increased moisture content on the chemical and biological stability of lipoplexes containing additives. Our results show that both ROS and TBARS are generated in lyophilized cakes during storage, and that agents such as DTPA or alpha-tocopherol are efficient in protecting lipid/DNA complexes against oxidative damage in the dried state. Our experiments also indicate that higher residual moisture has a deleterious effect on the stability of lipid/DNA complexes during storage.

Metal contaminants promote degradation of lipid/DNA complexes during lyophilization.

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.

Low molecular weight dextrans stabilize nonviral vectors during lyophilization at low osmolalities: concentrating suspensions by rehydration to reduced volumes.

Stabilization of nonviral vectors during freezing and drying requires formulation with protective excipients such that transfection rates and physical characteristics are maintained upon reconstitution. While many studies have demonstrated the ability of disaccharides (e.g., sucrose) to effectively protect nonviral vectors during lyophilization, the sucrose/DNA weight ratios required to achieve stability result in formulations that are not osmotically compatible with the subcutaneous (SC) or intramuscular (IM) injection of a typical dose of plasmid DNA. In an effort to reduce the formulation osmolality, dextrans possessing a range of molecular weights were investigated for their ability to serve as protectants. Dextran 3000 proved to be the most effective of the dextrans tested, and offered similar protection to sucrose on a weight basis. However, the advantage of employing this excipient is that the resulting osmolality is reduced by approximately 40% as compared to an equivalent weight of sucrose. Moreover, the use of dextran allows lyophilized vector preparations to be rehydrated to reduced volumes, essentially concentrating vectors prior to administration. Utilizing a combination of dextran 3000 and sucrose, we demonstrate that complexes of polyethylenimine (PEI) and DNA lyophilized at 0.1 mg/mL can be concentrated tenfold upon rehydration, resulting in an isotonic formulation containing 1 mg/mL DNA that can provide more realistic injection volumes for animal studies, and is compatible with clinical trials involving SC and IM injection.

The stability of lyophilized lipid/DNA complexes during prolonged storage.

It is well known that excipients are required to protect nonviral vectors during the lyophilization process. The goal of this study is to describe the stability of lyophilized nonviral vector preparations on pharmaceutically relevant timescales and provide insight into the factors that govern long-term stability of vectors in the dried state. Lipid/DNA complexes were lyophilized in glucose, sucrose, or trehalose and stored for a period of up to 2 years at five different temperatures (-20, 4, 22, 40, 60 degrees C). We evaluated simultaneously the physico-chemical characteristics (size, zeta potential, ethidium bromide (EtBr) accessibility, supercoiled DNA content) and the ability of vector formulations to transfect COS-7 cells at different time intervals. In addition, a fluorescence assay was utilized to assess levels of ROS in the dried cake after storage. The physical state of each formulation was evaluated by determination of the glass transition temperature and residual moisture content, before and after storage. Results from our stability study show that a progressive degradation of lipid/DNA complexes occurs in terms of transfection rates, particle size, dye accessibility, and supercoil content, even when samples are stored at low temperatures (e.g., -20 degrees C). Furthermore, our preliminary results on the quantification of free radicals in rehydrated formulations emphasize the importance of developing strategies to prevent the formation of reactive oxygen species (ROS) during prolonged storage in the dried state.