title: 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.
abstract: The aim of the study was to comprehensively investigate the influence of the freezing step during lyophilization on the stability of gene-delivery particles in order to better understand particle stabilization during freezing. Particle size of plasmid/linear polyethylenimine (LPEI) polyplexes at two DNA concentrations and at increasing sucrose-DNA ratios was investigated separately as a function of freezing procedure, ice-nucleation temperature, residence time of the particles in a partially frozen state, or incomplete freezing. Using a numerical model, the increase in sucrose concentration and system viscosity and corresponding bimolecular reaction rates were theoretically estimated. Freezing with a temperature-hold step after ice nucleation negatively influenced particle stability. Ice-nucleation temperature had an impact only at low DNA concentrations. Particle stability was highly reduced during the early part of freezing (<-3°C), especially at low shelf-ramp rates. In this phase, bimolecular reaction rates increase greatly at still low system viscosity. Below a critical temperature (≤∼-18°C) and at high system viscosity, no further particle aggregation occurred. In conclusion, the initial sample viscosity rather than the unfrozen volume and the residence time of particles in the low-viscosity state are the predominant factors in particle stabilization, which likely apply to aggregation in any system.
authors.affiliation: Department of Pharmacy, Pharmaceutical Technology and Biopharmaceutics, Ludwig-Maximilians-Universitaet, Munich 81377, Germany. julia.kasper@cup.uni-muenchen.de
authors.forename: Julia C
authors.initials: JC
authors.lastname: Kasper
authors.forename: Michael J
authors.initials: MJ
authors.lastname: Pikal
authors.forename: Wolfgang
authors.initials: W
authors.lastname: Friess
mesh_terms: D003460:Crystallization; D004247:DNA / Q000737:chemistry*; D005612:Freeze Drying / Q000379:methods*; D005615:Freezing; D007053:Ice / Q000032:analysis*; D008956:Models, Chemical; D010957:Plasmids / Q000737:chemistry*; D011094:Polyethyleneimine / Q000737:chemistry*; D013395:Sucrose / Q000737:chemistry; D014783:Viscosity
chemical_list: D007053:Ice; D013395:Sucrose; D011094:Polyethyleneimine; D004247:DNA