Effects of Moisture Content on the Storage Stability of Dried Lipoplex Formulations

Abstract

The purpose of this study is to investigate the effects of moisture content on the storage stability of freeze-dried lipoplex formulations. DC-Cholesterol: DOPE (dioleoyl phosphatidylethanolamine) / plasmid DNA lipoplexes were prepared at a 3-to-2 DC-Cholesterol + to DNA − molar ratio and lyophilized prior to storing at room temperature, 40 °C, and 60 °C for three months. Different residual moistures (1.93%, 1.10%, 1.06% and 0.36%) were obtained by altering the secondary drying temperatures. In addition to moisture content, lipoplex formulations were evaluated after freezedrying and/ or storage for particle size, transfection efficiency, accumulation of TBARS (thiobarbituric reactive substances), glass transition temperature, DNA supercoil content, and surface area. Lipoplex formulations stored at room temperature for 3 months maintain TBARS concentrations and supercoil contents. At higher storage temperatures, formulations possessing the highest moisture content (1.93%) maintained significantly lower TBARS concentrations and higher supercoil content than those with the lowest (0.36%) moisture content. Curiously, the intermediate moisture contents exhibited marked differences in stability despite virtually identical moisture contents. Subsequent measurements of surface area indicated that the lower stability corresponded to higher surface area in the dried cake, suggesting that there may be an interplay between water content and surface area that contributes to storage stability.

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

“…Therefore, we think that the observed particle size increases in our studies were most likely due to physicochemical changes in lipoplexes that promote aggregation upon rehydration [183]. However, despite the maintenance of particle size in many formulations (see Table 2), our previous reports have shown that progressive vector degradation occurred in spite of the high T g values and low moisture contents, indicating that mechanisms other than aggregation are responsible for the loss of biological activity during storage [178,179,183,276]. It is important to mention that under the experimental conditions of prolonged storage (e.g., 24 months), dried lipoplexes for- DTPA diethylenetriaminepentaacetic acid, M months, TBARS thiobarbituric acid reactive substances, w/w weight to weight ratio, DEPC Diethylpyrocarbonate Table 2 Storage studies of lyophilized ( LF) lipid-based therapeutics mulated with glucose also exhibited severe physical degradation (i.e., brown cake) indicative of nonenzymatic glycosylation via the Maillard reaction [183].…”

“…As a number of studies have implicated maintenance of particle size as a critical factor for the recovery of transfection activity [83,90,91,95,99,103,120,181,193], our recent results have demonstrated a relationship between retention of particle size and T g of the glassy excipient phase during prolonged storage [183]. This is clearly illustrated by the fact that lyophilized vectors stored at room temperature in trehalose (high T g ; [178,183,276,279]) show less tendency to aggregate as compared to glucose formulations (lower T g s; [183]). These findings are consistent with the idea that vectors possessing more restricted mobility are less prone to aggregation [127].…”

“…The effect of moisture content on the chemical and biological stability of lipid/ DNA complexes during storage has been recently explored by our group and others [178,276,279]. Considering that water is a potential source of ROS [191,286] and that it can participate in hydrolytic reactions that can chemically degrade biomolecules during storage [138,229], it is typically assumed that lower water contents might enhance the stability of lipoplexes in the dried state.…”

“…Despite this great progress, prolonged storage of lipoplexes in aqueous formulation yet is difficult to achieve considering the high sensitivity of both DNA and lipid components to hydrolytic and oxidative degradation [61,66,67,79,[271][272][273]. Furthermore, it has been reported that DNA functionality and structure may be damaged directly by co-oxidation with lipids [274][275][276]. In the last decade, there was a growing consensus that lyophilization technology is a suitable approach to stabilize batches of nonviral gene delivery systems as dehydrated formulations.…”

“…We found that in spite of the inclusion of demetalation and headspace oxygen displacement steps, formation of ROS was still observed in dried formulations, which suggests that these approaches are not sufficient to protect lipoplexes. However, the inclusion of a metal ion chelator (DTPA, 200 μM) or a lipid-soluble antioxidant (α-tocopherol, 50 μM) to our formulations prior to lyophilization enhanced the stability of dried lipoplexes during storage [178]; formulation strategies that have been applied in subsequent lipid-related storage studies [276].…”