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The stabilizing effect of mannitol during the freeze-drying of proteins was studied using L-lactate dehydrogenase (LDH, rabbit muscle), beta-galactosidase (Escherichia coli) and L-asparaginase (Erwinia chrysanthemi) as model proteins. Crystallization of mannitol was studied by powder X-ray diffraction and differential scanning calorimetry (DSC), in relation to the stabilizing effect. All the enzymes were protected concentration-dependently by amorphous mannitol, but the stabilizing effect was decreased with an increase in mannitol crystallinity. The heat-treatment of frozen solutions above crystallization temperature prior to drying enhanced mannitol crystallization and LDH inactivation. The importance of maintaining excipients in an amorphous state during freeze-drying, previously reported for Aspergillus oryzae beta-galactosidase (K. Izutsu et al., Pharm. Res., 10, 1233 (1993)), was confirmed using three different enzymes.

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The effects of amphiphilic excipients on the inactivation of lactate dehydrogenase (LDH) during freeze-thawing and freeze-drying were studied. Some amphiphilic excipients such as hydroxypropyl-beta-cyclodextrin (HP-beta-CD), CHAPS, polyethylene glycol (PEG) 3350, and sucrose fatty acid monoester prevented LDH inactivation during freeze-thawing and freeze-drying at a lower concentration than sugars and amino acids. Polyoxyethylene 9 lauryl ether and PEG 400 protected LDH during freeze-thawing but not during freeze-drying. The buffer concentration of the solution to be freeze-dried (10, 50, and 200 mM) affected the stabilizing effect of trehalose, but not that of HP-beta-CD. (c) 1994 John Wiley & Sons, Inc.

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Exosomes mediate communication between cells in the body by the incorporation and transfer of biological materials. To design an artificial liposome, which would mimic the lipid composition and physicochemical characteristics of naturally occurring exosomes, we first studied the physicochemical properties of exosomes secreted from HepG2 cells. The exosome stiffness obtained by atomic force microscopy was moderate. Some liposomes were then fabricated to mimic the representative reported lipid composition of exosomes. Their physicochemical properties and cellular internalization efficiencies were investigated to optimize the cellular internalization efficiency of the liposomes. A favorable internalization efficiency was obtained by incubating HeLa cells with 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC)/cholesterol (Chol)/1,2-dioleoyl-sn-glycero-3-phospho-l-serine (DOPS) (40/40/20 mol %) liposomes, which have a similar stiffness and zeta potential to exosomes. A dramatic increase in internalization efficiency was demonstrated by adding DOPS to simple DSPC/Chol liposomes. We found that DOPS had a more desirable effect on cellular internalization than its saturated lipid counterpart, 1,2-distearoyl-sn-glycero-3-phospho-l-serine. Furthermore, it was shown that the phosphatidylserine-binding protein, T-cell immunoglobulin mucin protein 4, was largely involved in the intracellular transfer of DSPC/Chol/DOPS liposomes. Thus, DOPS was a key lipid to provide the appropriate stiffness, zeta potential, and membrane surface affinity of the resulting liposome. Our results may help develop efficient drug carriers aiming to internalize active substances into cells.

43Freeze-drying is a popular method of ensuring the stability of proteins that are not stable enough in aqueous solutions during the period required for storage and distribution. 1,2) Various freeze-dried protein formulations contain excipients (e.g., sugars, polymers, and amino acids) that protect proteins from physical and chemical changes. Disaccharides (e.g., sucrose, trehalose) are the most popular among them because they stabilize proteins both thermodynamically and kinetically in aqueous solutions and freeze-dried solids. [3][4][5] The development of freeze-dried protein formulations containing amino acids is often more challenging than the development of formulations with saccharides because of the varied physical and chemical properties (e.g., crystallinity, glass transition temperature) of the freeze-dried amino acids, as well as their tendency to form complexes with other ingredients. 6) Many amino acids are considered to protect proteins basically in similar mechanisms with disaccharides. They thermodynamically stabilize protein conformation in aqueous solutions and probably in frozen solutions by being preferentially excluded from the immediate surface of proteins. 7) Glass-state amorphous solids formed by freeze-drying of the disaccharides or some amino acids protect proteins from structural changes thermodynamically by substituting surrounding water molecules. 8) They also reduce chemical degradation of freeze-dried proteins kinetically by reducing the molecular mobility. 2,8) In addition, some amino acids (e.g., L-arginine) also prevent protein aggregation in aqueous solutions prior to the drying process and after reconstitution. 9) Choosing appropriate counterions that form glass-state solid should be one of the key factors in designing amino acid-based amorphous freeze-dried formulations. 10,11) For example, glass transition temperatures (T g ) of freeze-dried Lhistidine salts depend largely on the counterions. 12) Colyophilization of L-arginine and multivalent inorganic acids (e.g., H 3 PO 4 , H 2 SO 4 ) results in glass-state amorphous solids that protect proteins during the process and storage (e.g., tissue plasminogen activator formulation, PDR 2003). 13) Some organic acid and inorganic cation combinations (e.g., sodium citrates) also form high glass transition temperature amorphous solids. 14) Various functional groups (e.g., amino, carboxyl, hydroxyl) in the constituting molecules contributes significantly to form the glass-state amorphous salt solids. 15) Producing glass-state amorphous solids by freeze-drying of amino acid and organic acid combinations, and their application in pharmaceutical formulations are interesting topics to explore. 15) The purpose of this study was to produce stable amorphous solids that protect proteins by freeze-drying combinations of amino acids and organic acids. The physical properties of frozen solutions and freeze-dried solids containing the popular excipients and model chemicals were studied. The effect of the excipient combinations on the freeze-drying ...