Synergistic effects of surfactants and sugars on lipoplex stability during freeze-drying and rehydration

Abstract

The stability of non-viral vectors during freeze-drying has been well-studied, and it has been established that sugars can protect lipoplexes during freeze-drying. However low levels of damage are often observed after freeze-drying, and this damage is more evident in dilute lipoplex preparations. By investigating the stability of lipoplexes after each step in the freeze-drying cycle (i.e., freezing, primary drying, and secondary drying) we strive to understand the mechanisms responsible for damage and identify improved stabilization strategies. N-(1-(2,3-dioleoyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTAP)-cholesterol/plasmid DNA lipoplexes were prepared at an equimolar DOTAP to cholesterol ratio, and a 3-to-1 DOTAP + to DNA − charge ratio. Our experiments indicate that despite sufficient levels of "stabilizing" sugars, significant damage is still evident when dilute lipoplex preparations are subjected to freeze-drying. Analysis of the different stages of freeze-drying suggests that significant damage occurs during freezing, and that sugars have a limited capacity to protect against this freezing-induced damage. Similar effects have been observed in studies with proteins, and surfactants have been employed in protein formulations to protect against surface-induced damage, e.g., at the ice crystal, solid, air or sugar glass surfaces. However, the use of surfactants in a lipid-based formulation is inherently risky due to the potential for altering/solubilizing the lipid delivery vehicle. Our data indicate that judicious use of surfactants can reduce surface-induced damage, and result in better preservation of lipoplex size and transfection activity after freeze-drying.

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

“…Some of our most recent work has demonstrated the stabilizing effects of incorporating surfactants into lipoplex formulations during acute lyophilization stress. 44 The current study included an investigation of the effects of Tween 80 (40 µM) on the storage stability of lipoplex formulations, but we observed that oxidation during storage was dramatically increased in the presence of Tween 80 (data not shown). We suspect that the rapid deterioration of samples formulated with Tween 80 was due to the tendency of this unsaturated detergent to participate in oxidation, and future studies will investigate this hypothesis.…”

PepFect 14, a novel cell-penetrating peptide for oligonucleotide delivery in solution and as solid formulation

“…There was no statistically significant loss in transfection efficiencies at any time-point except for the 60°C 8-weeks point where the transfection efficiency decreased to 70% of the initial value (Figure 8). Compared to lyophilized lipoplexes (27,28), PF14 formulations demonstrated an excellent stability profile without further additives or special storage conditions.

Figure 8.Stability testing.

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Investigations on Polyplex Stability During the Freezing Step of Lyophilization Using Controlled Ice Nucleation—The Importance of Residence Time in the Low-Viscosity Fluid State

“…Analogously, Allison et al 7 stated that the reduced stability of lipoplexes in more dilute samples could be explained in general by an increased interaction with the ice-crystal surface area. This assumed interaction of the particles with the ice surface is additionally supported by Yu and Anchordoquy,40 who reported that only the synergistic effects of surfactants and sugars are capable to stabilize dilute polyplex formulations. In contrast to our study, Allison et al 7 could achieve complete preservation of particle size for more dilute lipoplex samples.…”