Mempro™ Liposome Preparation by Freeze-thawing and Freeze-drying

During the past decades, Creative Biostructure has devoted to the liposome research and application, and we have established an advanced Mempro™ Liposome platform to offer various products and services. Creative Biostructure is rich-experienced in liposome preparation services, mastering numbers of mechanical and non-mechanical techniques to perform custom production. Freeze-thawing and freeze-drying techniques are applied to produce and refine the liposomes with improved properties.

Figure 1. Internal sucrose concentrations have effect on cryoTEM morphology for liposomes before and after freeze–thaw. (D. Cipolla, et al., 2016)

Freeze-drying method

In freeze-drying method, appropriate water-soluble carriers such as sucrose, mannose, and lactose can be used as lyoprotectants for lyophilization of liposomal dispersions. Together with the water-soluble carriers, the liposome preparation system is sterilized by filtration and forced to freeze-drying.

We can either store the subsequent lyophilized liposome powder indefinitely or rehydrate the liposome powder with appropriate aqueous buffer to further generate a spontaneous liposomal colloidal suspension.

The existing/preformed liposomes, which have been subjected to dehydration-rehydration cycles, are adopted to form the freeze-dried rehydrated liposomes. Lyoprotectants act as the key factor to help liposomes adopt proper configuration after freeze-drying, avoiding liposome (especially neutral charged liposomes) aggregation or leakage of the encapsulated agents. Adding slight negative charge to liposomal system is another approach to prevent liposome aggregation during rehydration. The lipid bilayers of liposomes prepared by freeze-drying method and the encapsulated agents are in close contact, therefore, high encapsulation efficiencies can be achieved.

Freeze-thawing method

Multilamellar vesicle (MLV) has a low encapsulation capacity since it is quite heterogeneous both in size and lamellarity. Freeze-thawing method is usually applied to increase the encapsulation capacity of MLV by decreasing its lamellarity. Freeze-thawing method is performed by submerging liposome sample under water and changing temperatures with large gradients.

Elimination of the solutes from the ice phase during freezing leads to the concentrated entrapment in the residual fluid. Electrostatic interaction between the lipids with negative charge and the solution with positive charge results in vesicle formation. Liposome can be subjected to multiple freeze-thawing cycles at a temperature above the phase transition temperature (Tm) to further improve the encapsulation efficiency. We should also note that increasing the phospholipid concentration or increasing the ionic strength of the medium would strongly inhibit this type of preparation.

Besides freeze-thawing and freeze-drying method, Creative Biostructure also provides various methods such as thin-film, sonication, extrusion, and reverse phase evaporation to prepare custom liposomes. Creative Biostructure is a reliable and professional business associate to clients in academic or industrial field. Please feel free to contact us for a detailed quote.

References:
C. V. Kulkarni, et al. (2016). Effect of fullerene on the dispersibility of nanostructured lipid particles and encapsulation in sterically stabilized emulsions. Journal of Colloid and Interface Science, 480(15): 69-75.
N. J. Alves, et al. (2016). Protecting enzymatic function through directed packaging into bacterial outer membrane vesicles. Scientific Reports, 6: 24866.
D. Cipolla, et al. (2016). Formation of drug nanocrystals under nanoconfinement afforded by liposomes. RSC Advances, 6: 6223-6233.