Amino acids as cryoprotectants for liposomal delivery systems

Liposomes provide an efficient delivery system for solubilisation and delivery of both small and macro molecules. However, they suffer from the disadvantage of instability when stored as aqueous dispersions. Cryoprotection of the liposomal systems provides an effective approach to overcome poor stability whilst maintaining formulation characteristics, although, the formulation of a freeze-dried product requires the consideration of not only the selection of an appropriate cryoprotectant, but also needs careful consideration of the processing parameters including pre-freezing conditions, primary and secondary drying protocols along with optimisation of cryoprotectant concentration. This current work investigates the application of amino acids as potential cryoprotectants for the stabilisation of liposomes, and results indicate that amino acids show biphasic nature of stabilisation with 4 mol of cryoprotectant per mole of the lipid exhibiting optimum cryoprotection. The investigations of process parameters showed that the pre-freezing temperatures below the glass transition of the amino acids followed by drying for over 6 h resulted in stable formulations. Studies investigating the efficiency of drug retention showed that the cryoprotection offered by lysine was similar to that shown by trehalose, suggesting that amino acids act as effective stabilisers. ESEM analysis was carried out to monitor morphology of the rehydrated liposomes. © 2007 Elsevier B.V. All rights reserved.

The effect of Me2SO overexposure during cryopreservation on HOS TE85 and hMSC viability, growth and quality

With the cell therapy industry continuing to grow, the ability to preserve clinical grade cells, including mesenchymal stem cells (MSCs), whilst retaining cell viability and function remains critical for the generation of off-the-shelf therapies. Cryopreservation of MSCs, using slow freezing, is an established process at lab scale. However, the cytotoxicity of cryoprotectants, like Me2SO, raises questions about the impact of prolonged cell exposure to cryoprotectant at temperatures >0 °C during processing of large cell batches for allogenic therapies prior to rapid cooling in a controlled rate freezer or in the clinic prior to administration. Here we show that exposure of human bone marrow derived MSCs to Me2SO for ≥1 h before freezing, or after thawing, degrades membrane integrity, short-term cell attachment efficiency and alters cell immunophenotype. After 2 h's exposure to Me2SO at 37 °C post-thaw, membrane integrity dropped to ∼70% and only ∼50% of cells retained the ability to adhere to tissue culture plastic. Furthermore, only 70% of the recovered MSCs retained an immunophenotype consistent with the ISCT minimal criteria after exposure. We also saw a similar loss of membrane integrity and attachment efficiency after exposing osteoblast (HOS TE85) cells to Me2SO before, and after, cryopreservation. Overall, these results show that freezing medium exposure is a critical determinant of product quality as process scale increases. Defining and reporting cell sensitivity to freezing medium exposure, both before and after cryopreservation, enables a fair judgement of how scalable a particular cryopreservation process can be, and consequently whether the therapy has commercial feasibility.