hydroxyethyl starch) are relatively high compared to that of glycerol
[47] (see Fig. 1c). Compounds with ice blocking properties may be
used to limit re-crystallization and its associated damage during the
warming process.
4.2 Lyoprotective
Agents
Lyoprotective agents generally protect during both freezing and
drying and can be considered as a subset of the group of CPAs.
Non-reducing disaccharides have been widely used as lyoprotective
agents for freeze-drying of proteins and liposomal systems. Two
main theories exist on how sugars protect liposomes during drying.
The water replacement hypothesis (see Fig. 2c, d) postulates that
sugars replace the water surrounding the phospholipid head groups
during drying, by replacing the hydrogen bonding interactions
between water and the head groups thereby preventing that membrane acyl chains come in closer proximity of each other. This
prevents membranes from undergoing a liquid-crystalline to
gel-phase transition and its associated membrane leakiness
[2]. Alternatively, it has been postulated that sugars protect lipid
membranes by trapping the water molecules surrounding the lipid
head groups [48]. Both theories are not mutually exclusive, and
each may play a role during different phases of the drying process.
For example, during the initial phase of drying, water entrapment
may prevail, whereas water replacement would take place after
prolonged drying or when supplying heat to the system [49]. Moreover, disaccharides reduce the hydration forces acting between lipid
bilayers in solution or upon freezing and drying. Solutes may affect
hydration forces either if they are adsorbed onto the membranewater interface or when excluded from the interface [50].
In contrast with typical CPAs such as glycerol or DMSO, sugars
can form a glassy state at room temperature, which is an important
protective feature for stable storage in the dried state (see Fig. 1c).
As described above, a glass is a highly viscous state which is formed
below a characteristic T g . Biomolecules (or cellular structures) are
embedded in a glassy matrix, while simultaneously molecular
mobility and damaging reactions are slowed down [15, 51,
52]. Among the disaccharides, trehalose has a T g which is nearly
60
C higher than that of sucrose which has the same molecular
weight [8]. Macromolecules, such as albumin and hydroxyethyl
starch (HES), can be added to freeze-drying formulations to
increase the T g and storage stability [53, 54]. Water acts as a
plasticizer and decreases T g of (freeze-)dried samples. T g and
molecular mobility in the glassy state, and hence sample storage
stability, are dependent on the residual moisture content after
freeze-drying [55].
Principles of Cryopreservation and Freeze-Drying
11
[47] (see Fig. 1c). Compounds with ice blocking properties may be
used to limit re-crystallization and its associated damage during the
warming process.
4.2 Lyoprotective
Agents
Lyoprotective agents generally protect during both freezing and
drying and can be considered as a subset of the group of CPAs.
Non-reducing disaccharides have been widely used as lyoprotective
agents for freeze-drying of proteins and liposomal systems. Two
main theories exist on how sugars protect liposomes during drying.
The water replacement hypothesis (see Fig. 2c, d) postulates that
sugars replace the water surrounding the phospholipid head groups
during drying, by replacing the hydrogen bonding interactions
between water and the head groups thereby preventing that membrane acyl chains come in closer proximity of each other. This
prevents membranes from undergoing a liquid-crystalline to
gel-phase transition and its associated membrane leakiness
[2]. Alternatively, it has been postulated that sugars protect lipid
membranes by trapping the water molecules surrounding the lipid
head groups [48]. Both theories are not mutually exclusive, and
each may play a role during different phases of the drying process.
For example, during the initial phase of drying, water entrapment
may prevail, whereas water replacement would take place after
prolonged drying or when supplying heat to the system [49]. Moreover, disaccharides reduce the hydration forces acting between lipid
bilayers in solution or upon freezing and drying. Solutes may affect
hydration forces either if they are adsorbed onto the membranewater interface or when excluded from the interface [50].
In contrast with typical CPAs such as glycerol or DMSO, sugars
can form a glassy state at room temperature, which is an important
protective feature for stable storage in the dried state (see Fig. 1c).
As described above, a glass is a highly viscous state which is formed
below a characteristic T g . Biomolecules (or cellular structures) are
embedded in a glassy matrix, while simultaneously molecular
mobility and damaging reactions are slowed down [15, 51,
52]. Among the disaccharides, trehalose has a T g which is nearly
60
C higher than that of sucrose which has the same molecular
weight [8]. Macromolecules, such as albumin and hydroxyethyl
starch (HES), can be added to freeze-drying formulations to
increase the T g and storage stability [53, 54]. Water acts as a
plasticizer and decreases T g of (freeze-)dried samples. T g and
molecular mobility in the glassy state, and hence sample storage
stability, are dependent on the residual moisture content after
freeze-drying [55].
Principles of Cryopreservation and Freeze-Drying
11