The Main Function of Yingtai Freeze-Drying Protectants

The Main Function of Yingtai Freeze-Drying Protectants

The primary function of freeze-drying protectants is to reduce damage to liposomes during freezing and thawing processes. Commonly studied protectants include monosaccharides, disaccharides, oligosaccharides, polysaccharides, polyols, and other water-soluble polymers. The protective mechanisms of these agents vary. For instance, mannitol can increase viscosity, reduce the crystallization rate of water, and lower the eutectic melting temperature of internal aqueous phases; D-glucose can concentrate in the ice crystal growth spaces during freezing, forming a protective layer over liposomes that prevents ice crystals from embedding in the lipid bilayer; and alginate stabilizes the dehydrated membrane during freezing through hydrogen bonding with the polar groups of phospholipids, effectively replacing residual water around these polar groups while increasing the spatial effect and lowering phase transition temperatures. The typical usage of protectants is around 2% to 5% (w/v). The protective effects of freeze-drying agents on liposomes depend on factors such as their concentration, freeze-drying processes, and the properties of liposomes and drugs.

Carbohydrate Protectants

Carbohydrate substances are generally used as protectants for liposome freeze-drying. These protectants provide several protective effects on liposomes during freezing:

1. Preventing Fusion

Protectants reduce the contact and adhesion between vesicles, thereby preventing fusion. As the liposome suspension freezes and ice crystals grow, the protectant gradually concentrates and distributes around the liposome vesicles, acting as a spacer to inhibit vesicle fusion and drug leakage.

2. Inhibiting Ice Crystal Growth

Protectants can suppress ice crystal growth, decreasing the likelihood of ice crystals embedding in the lipid bilayer and reducing damage to liposome vesicles, thereby preventing membrane rupture.

3. Increasing Transition Temperature

Protectants can raise the glass transition temperature of the liposome suspension, allowing for partial vitrification under certain cooling rates. This avoids crystallization and reduces various damages caused by ice crystal growth.

4. Enhancing Viscosity

During the freezing of the liposome suspension, carbohydrate protectants increase the viscosity of the solution, thereby weakening the crystallization of water and achieving the desired protective effect.

5. Forming Hydrogen Bonds

Freeze-drying protectants can form hydrogen bonds with the polar groups of liposome phospholipids, replacing water as a stabilizer after dehydration, maintaining the integrity of the liposome membrane, and inhibiting drug leakage. This mechanism is referred to as the "water replacement hypothesis." Without freeze-drying protectants, the phase transition temperature (Tm) of freeze-dried liposomes can increase significantly. However, with the addition of carbohydrate protectants, the lost water can be replaced in the polar regions of the membrane interface, avoiding drastic changes in Tm. Depending on the freeze-drying conditions, Tm can be higher or lower than the phase transition temperature (Tc) of hydrated liposomes. The stronger the interaction between sugars and phospholipids, the lower Tm becomes, leading to stronger protective effects. The extent of Tm reduction correlates well with the stability of the freeze-dried product.

Conditions for Protectants

In summary, during the drying process of liposomes, protectants must meet two basic conditions: 1. They should have a relatively high glass transition temperature (Tg'), which is a stringent requirement. 2. They must interact well with the lipid bilayer. Mixed protectants also need to satisfy these criteria. For example, using glucose or hydroxyethyl starch (HES) alone as a protectant can lead to instability in egg phosphatidylcholine liposomes during freeze-drying, but combining them can yield better results. In the freeze-drying process, they play different roles: glucose interacts with phospholipids, while HES does not interact with the lipid bilayer but provides a higher Tg'. The combination can also be applied for freeze-drying red blood cells.