Why Is Trehalose Such A Good Cryoprotectant?

Many factors in the freeze-drying of foods, medicines, and organisms (for example, chemical composition, freezing rate, freezing and dehydration stress, glass transition temperature, residual moisture in dry solids, etc.) will affect the stability of the active components. In order to protect the quality and activity of biological products and some special preparations such as liposomal drugs, we usually add cryoprotectant agents to pharmaceutical formulations and then prepare them into lyophilized preparations. For the freeze-drying protection mechanism, there are two main hypotheses at present. And the more recognized one is water replacement hypothesis.

Trehalose Cryoprotectant

Freeze-drying protection mechanism — water replacement hypothesis

Many researchers believe that due to a large number of hydrogen bonds in protein molecules, bound water is connected to protein molecules through hydrogen bonds. When the protein loses water, the hydroxyl group of the cryoprotectant agents can replace the hydroxyl group of the water on the surface of the protein, so that the protein surface forms a presumed hydration film, which can protect the hydrogen bonding position from being directly exposed to the surrounding environment, thus stabilizing the high-level structure of proteins, preventing denaturation and maintaining the integrity of protein structure and function even in the case of freezing and drying at low temperature.

Requires for cryoprotectant agents

The theoretical basis of the two hypotheses is that the chemical liquid has achieved partial or full vitrification freezing. Therefore, suitable cryoprotectant agents need to have the following four characteristics: high glass transition temperature, poor water absorption, low crystallization rate, and no reducing group.

Why is trehalose such a good cryoprotectant?

Experiments have proved that monosaccharides (such as glucose, galactose, etc.) cannot protect proteins during the freeze-drying process, because monosaccharides can only provide a weak stabilizing effect, causing irreversible denaturation of proteins before they are dehydrated. Nonreducing disaccharides (sucrose, trehalose) are often used as cryoprotectant agents of many biological products. This is because the disaccharide can not only act as a low-temperature protective agent, but also a drying protectant. And the absence of reducing groups will not cause the biological product to undergo protein browning reaction which may deteriorate and inactivate the proteins. Therefore, in the freeze-drying process of biological drugs, sucrose and trehalose are the two most commonly used cryoprotectant agents.

Trehalose cryoprotectant vs. sucrose cryoprotection

Compared with sucrose, trehalose has a higher glass transition temperature (120 ℃), so the trehalose cryoprotectant is less likely to form ice crystals. And trehalose also has a magical hydration capacity: the number of non-frozen water molecules around trehalose per glucose unit is the largest number among sugars. Trehalose cryoprotectant can form a more rigid trehalose/water structure and has stronger anti-freeze ability. This is why the trehalose cryoprotectant is better than sucrose cryoprotection.

Trehalose cryoprotectant vs. glucose

In addition, trehalose is also a good liposome cryoprotectant. Studies have shown that the lyophilized liposomes with trehalose as the protective agent have the smallest particle size change and the best protection effect; while the particle size change using glucose is the largest and the protection effect the worst. A 10% concentration of trehalose cryoprotectant provides the best protection for lyophilized liposomes.

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