Synergistic effects of surfactants and sugars on lipoplex stability during freeze-drying and rehydration.
The stability of nonviral 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-cholesterol ratio, and a 3:1 DOTAP(+)-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, for example, 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.
Yu
Jinxiang
J
School of Pharmacy, University of Colorado Denver, 4200 E. Ninth Ave., Denver, Colorado 80262, USA. jinxiang.yu@uchsc.edu
Anchordoquy
Thomas J
TJ
D000818:Animals; D019556:COS Cells; D002241:Carbohydrates / Q000737:chemistry*; D002522:Chlorocebus aethiops; D002784:Cholesterol / Q000737:chemistry; D004247:DNA / Q000008:administration & dosage* / Q000235:genetics; D005612:Freeze Drying; D008055:Lipids / Q000737:chemistry*; D010316:Particle Size; D000644:Quaternary Ammonium Compounds / Q000737:chemistry; D013501:Surface-Active Agents / Q000737:chemistry*; D014162:Transfection
D002241:Carbohydrates; D008055:Lipids; D000644:Quaternary Ammonium Compounds; D013501:Surface-Active Agents; D004247:DNA; D002784:Cholesterol
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