Role of freeze-drying in the presence of mannitol on the echogenicity of echogenic liposomes

Abstract

Echogenic liposomes (ELIPs) are an excellent candidate for ultrasound activated therapeutics and imaging. Although multiple experiments have established their echogenicity, the underlying mechanism has remained unknown. However, freeze-drying in the presence of mannitol during ELIP preparation has proved critical to ensuring echogenicity. Here, the role of this key component in the preparation protocol was investigated by measuring scattering from freshly prepared freeze-dried aqueous solution of mannitol-and a number of other excipients commonly used in lyophilization-directly dispersed in water without any lipids in the experiment. Mannitol, meso-erythritol, glycine, and glucose that form a highly porous crystalline phase upon freeze-drying generated bubbles resulting in strong echoes during their dissolution. On the other hand, sucrose, trehalose, and xylitol, which become glassy while freeze-dried, did not. Freeze-dried mannitol and other crystalline substances, if thawed before being introduced into the scattering volume, did not produce echogenicity, as they lost their crystallinity in the thawed state. The echogenicity disappeared in a degassed environment. Higher amounts of sugar in the original aqueous solution before freeze-drying resulted in higher echogenicity because of the stronger supersaturation and crystallinity. The bubbles created by the freeze-dried mannitol in the ELIP formulation play a critical role in making ELIPs echogenic.

In vitro characterization of sonothrombolysis and echocontrast agents to treat ischemic stroke

“…It has been postulated that mannitol causes defects in the lipid bilayer during freezing, which traps gas. However, the studies performed by Kumar and colleagues 52 demonstrate that mannitol nucleates bubbles (outgassing) by increasing the local concentration of sugar, which decreases the solubility of gas in the liquid. These investigators conjectured that bubbles nucleated both within the liposomes and in solution could account for the echogenicity of ELIP.…”

“…A recent report 52 showed that freeze-dried crystals of mannitol nucleated bubbles upon reconstitution in water, even without the presence of liposomes. Freeze-drying in the presence of mannitol, a cryoprotectant, is essential for producing echogenic liposomes 53 .…”

Echogenic exosomes as ultrasound contrast agents

“…The procedure was motivated by our as well as others' studies that indicated that mannitol plays a crucial role during the preparation of echogenic liposomes and polymersomes encapsulating gaseous cores that render such vesicles echogenic. 24,51,53 The echogenicity of the specially prepared exosomes were investigated using commercial ultrasound scanner both in vitro and in vivo. We also measure their scattered responses in a customized setup.…”

“…Kumar et al while exploring the role of freeze-dried mannitol on the echogenicity of liposomes and polymersomes also found strong echogenicity of freeze-dried mannitol powder by itself in DI water, which is further enhanced by addition of BSA. 57 It was hypothesized that BSA acts like a surfactant to the microbubbles stabilizing them and allowing them to remain in the solution longer. The present study seems to indicate a similar stabilizing role of BSA for the air pockets created in association with exosomes.…”

EphA2-Receptor Targeted PEGylated Nanoliposomes for the Treatment of BRAFV600E Mutated Parent- and Vemurafenib-Resistant Melanoma

“…Surprisingly, we found that mannitol in combination with trehalose has a positive effect on particle size of reconstituted liposomes. It was reported that the degree of mannitol crystallization during freeze drying can be controlled by processing conditions and chemical composition [50]. Differential scanning calorimetry (DSC) studies can be used for liposome quality control by thermal analysis to determine purity, polymorphic forms, and the melting point of the sample [51].…”