Yingtai Freeze-Drying Classroom: Common Freeze-Drying Protectants

Yingtai Freeze-Drying Classroom: Common Freeze-Drying Protectants

1. Monosaccharides

Common monosaccharides include glucose, mannitol, galactose, and fructose. Glucose provides good protection for the morphology, particle size, and encapsulation efficiency of liposomes during freeze-thaw processes, but its glass transition temperature (Tg') is relatively low, leading to significant drug leakage when used alone. Mannitol, galactose, and fructose have similar protective effects on liposomes as glucose.

2. Disaccharides

Disaccharides are the most studied and effective freeze-drying protectants, commonly including trehalose, maltose, sucrose, and lactose. Among these, trehalose is a non-reducing disaccharide that protects membrane functions under anhydrous conditions and maintains normal calcium ion transport across membranes. It also helps liposomes retain their integrity during complete dehydration. Other disaccharides only exhibit good dehydration protection at concentrations higher than physiological levels. Research by Crowe et al. indicates that directly freeze-drying palmitoylphosphatidylcholine-phosphatidylserine (PS) liposomes results in significant or complete leakage of water-soluble drugs. However, adding appropriate amounts of trehalose to the inner and outer aqueous phases of liposomes before freeze-drying allows for over 95% retention of drugs after rehydration. It was also found that the concentration of trehalose needed to inhibit liposome fusion is much lower (less than 1/10) than that required to prevent drug leakage. Direct freeze-drying can lead to aggregation and increased particle size of egg yolk phosphatidylcholine (EPC) liposomes, but using trehalose as a protectant maintains particle size without significant change upon rehydration. Trehalose, maltose, and sucrose provide similar freeze-drying protection for dipalmitoylphosphatidylcholine (DPPC) liposomes, with about 90% retention of water-soluble drugs after rehydration, significantly better than glucose, galactose, and mannitol. Van Winden et al. prepared doxorubicin freeze-dried liposomes using trehalose, sucrose, lactose, and maltose as protectants, achieving over 90% drug retention without significant changes in particle size after rehydration. By covalently bonding sucrose with fatty acids to create fatty acid sucrose esters, the stability of EPC and hydrogenated egg yolk phosphatidylcholine liposomes during freeze-thaw and rehydration processes was assessed. Results showed that using fatty acid sucrose esters alone could not prevent leakage of carboxyfluorescein. However, adding 5% sucrose increased carboxyfluorescein retention in liposomes to 95% after rehydration.

3. Oligosaccharides

Studies show that glucose provides weak protection against carboxyfluorescein leakage in dipalmitoylphosphatidylcholine (DOPC) and EPC liposomes, and can lead to some degree of liposome aggregation and fusion when used alone. However, it offers relatively high protection for DPPC liposomes, while maltose and maltotriose provide better protection for EPC liposomes. Similar studies indicate that using maltotriose as a protectant can achieve about 90% drug retention in freeze-dried liposomes after rehydration. The protective effect of other maltodextrins decreases with an increasing number of glucose residues. Cycloinulohexaose (CF-6) effectively inhibits liposome fusion and drug leakage during freeze-drying, with no significant changes in particle size distribution after rehydration, and retains over 85% of water-soluble drugs. Adding appropriate amounts of glycerol, ethylene glycol, propylene glycol, glycine, and proline can further enhance freeze-drying protection, achieving about 90% drug retention, possibly due to increased hydrogen bonding between lipids and CF-6.

4. Polyols

Tanaka et al. studied the freeze-drying protective effects of polyols such as sorbitol, myo-inositol, and mannitol on liposomes. The results indicated that polyols are less effective than disaccharides, with significant aggregation observed in freeze-dried products. Freeze-dried products using myo-inositol and sorbitol as protectants showed significant increases in particle size and severe drug leakage; nearly all drugs leaked from mannitol systems. However, mannitol’s higher melting point is often advantageous as a co-protectant to maintain the appearance of freeze-dried products.

5. Combination of Multiple Protectants

Using two or more protectants in combination can achieve better results than using a single one. Water-soluble polymers such as polyvinylpyrrolidone (PVP), PEG, polysaccharides, starch, and cellulose derivatives can also be combined with the aforementioned protectants. Combining fructooligosaccharides or hydroxyethyl starch with glucose can lead to over 85% drug retention after rehydration in freeze-dried EPC liposomes. When used alone, trehalose resulted in a 63% retention rate for freeze-dried methotrexate liposomes, which increased to over 70% when combined with PEG. Additionally, borate ions or inorganic phosphates can form complexes with trehalose in a dry state, enhancing trehalose’s Tg'.