Numerous organisms are capable of surviving more or less complete dehydration. A common feature in their biochemistry is that they accumulate large amounts of disaccharides, the most common of which are sucrose and trehalose. Over the past 20 years, we have provided evidence that these sugars stabilize membranes and proteins in the dry state, most likely by hydrogen bonding to polar residues in the dry macromolecular assemblages. This direct interaction results in maintenance of dry proteins and membranes in a physical state similar to that seen in the presence of excess water. An alternative viewpoint has been proposed, based on the fact that both sucrose and trehalose form glasses in the dry state. It has been suggested that glass formation (vitrification) is in itself sufficient to stabilize dry biomaterials. In this review we present evidence that, although vitrification is indeed required, it is not in itself sufficient. Instead, both direct interaction and vitrification are required. Special properties have often been claimed for trehalose in this regard. In fact, trehalose has been shown by many workers to be remarkably (and sometimes uniquely) effective in stabilizing dry or frozen biomolecules, cells, and tissues. Others have not observed any such special properties. We review evidence here showing that trehalose has a remarkably high glass-transition temperature (Tg). It is not anomalous in this regard because it lies at the end of a continuum of sugars with increasing Tg. However, it is unusual in that addition of small amounts of water does not depress Tg, as in other sugars. Instead, a dihydrate crystal of trehalose forms, thereby shielding the remaining glassy trehalose from effects of the added water. Thus under less than ideal conditions such as high humidity and temperature, trehalose does indeed have special properties, which may explain the stability and longevity of anhydrobiotes that contain it. Further, it makes this sugar useful in stabilization of biomolecules of use in human welfare.

Trehalose is a nonreducing disaccharide of glucose commonly found at high concentrations in anhydrobiotic organisms. In the presence of trehalose, dry dipalmitoyl phosphatidylcholine (DPPC) had a transition temperature similar to that of the fully hydrated lipid, whereas DPPC dried without trehalose had a transition temperature about 30 degrees Kelvin higher. Results obtained with infrared spectroscopy indicate that trehalose and DPPC interact by hydrogen bonding between the OH groups in the carbohydrate and the polar head groups of DPPC. These and previous results show that this hydrogen bonding alters the spacing of the polar head groups and may thereby decrease van der Waals interactions in the hydrocarbon chains of the DPPC. This interaction between trehalose and DPPC is specific to trehalose. Hence this specificity may be an important factor in the ability of this molecule to stabilize dry membranes in anhydrobiotic organisms.

We believe we have established the major principles governing the stabilization of living cells in the unique condition known as anhydrobiosis. These findings have permitted us to design ways to stabilize membrane vesicles, liposomes, and proteins, and perhaps eventually even intact cells that do not normally survive dehydration. In a complex phenomenon as ancient as anhydrobiosis, one would expect a myriad of adaptations to be required for survival of drying. But the arguments presented here suggest that a single perturbation--synthesis of a disaccharide such as trehalose or sucrose--is sufficient to achieve survival. We hasten to add, however, that it is now certain that additional adaptations are required; for instance, cells containing highly unsaturated lipids may survive drying for a short time, but they are so susceptible to degradation that they survive for a short time only. Thus the interpretation placed on the finding that trehalose can stabilize dry membranes must be regarded from this perspective as well. Nevertheless, we believe that the underlying physical principles governing stability of dry biological materials are universal.

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The microorganisms Escherichia coli DH5␣ and Bacillus thuringiensis HD-1 show an increased tolerance to freeze-drying when dried in the presence of the disaccharides trehalose and sucrose. When the bacteria were dried with 100 mM trehalose, 70% of the E. coli and 57% of the B. thuringiensis organisms survived, compared with 56 and 44%, respectively, when they were dried with sucrose. Only 8% of the E. coli and 14% of the B. thuringiensis organisms survived drying without the sugars. Fourier transform infrared spectroscopy was used to investigate the role of membrane phase transitions in the survival of the organisms during drying and rehydration. Both E. coli and B. thuringiensis showed an increase of 30 to 40؇C in the temperature of their phospholipid phase transition when dried without the sugars, while phase transition temperatures of those dried with the sugars remained near those of the hydrated cells. A Fourier transform infrared spectroscopy microscope made it possible to investigate the effects of drying on the protein structure in the intact cells. The amide II peak shifts from 1,543 cm ؊1 in the hydrated cells to about 1,533 cm ؊1 in the cells dried without sugar. There is no shift in the amide II peak when the cells are dried with trehalose or sucrose. We attribute the increased survival to the sugars' ability to lower the membrane phase transition temperature and to protect protein structure in the dry state. In addition to increasing the immediate survival of both species, the addition of trehalose protected the cells from the adverse effects of exposure to light and air while dry. E. coli dried with trehalose and exposed to light and air for 4 h had an increase in CFU of between 2,000 and 4,000 times the number obtained with E. coli dried without trehalose. B. thuringiensis showed an increase in CFU of 150% in samples dried with trehalose compared with samples dried without trehalose. The cells dried with sucrose did not show an increased tolerance to exposure following drying.

Simple sugars, especially disaccharides, stabilize biomaterials of various composition during air-drying or freeze-drying. We and others have provided evidence that direct interaction, an interaction that we believe is essential for the stabilization, between the sugar and polar groups in, for example, proteins and phospholipids occurs in the dry state. Some researchers, however, have suggested that the ability of the sugar to form a glass is the only requirement for stabilization. More recently, we have shown that both glass formation and direct interaction of the sugar and headgroup are often required for stabilization. In the present study, we present a state diagram for trehalose glass and suggest that the efficacy of this sugar for stabilization may be related to its higher glass transition temperatures at all water contents. We also show that trehalose and trehalose:liposome preparations form trehalose dihydrate as well as trehalose glass when rehydrated with water vapor. Formation of the dihydrate sequesters water, which might otherwise participate in lowering the glass transition temperature to below ambient. Because samples remain in the glassy state at ambient temperatures, viscosity is high and fusion between liposomes is prevented.

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