Isothermal Desiccation and Vitrification Kinetics of Trehalose−Dextran

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Isothermal Desiccation and Vitrification Kinetics of Trehalose-Dextran Solutions Alptekin Aksan* and Mehmet Toner Center for Engineering in Medicine and Department of Surgical Services, Massachusetts General Hospital, Harvard Medical School and Shriners Hospital for Children, 51 Blossom Street, Boston, Massachusetts 02114 Received August 18, 2003. In Final Form: April 13, 2004 The promise of dried state preservation is based on the hypothesis that lowering molecular mobility to halt chemical reaction and deterioration rates is the primary factor for the long-term stability of the dried specimen. In this research, the feasibility of utilizing isothermal, isobaric vitrification as an economical alternative to the preservation technologies currently in use (mainly, cryopreservation and lyophilization) is explored. Desiccation and vitrification kinetics of model trehalose and trehalose-dextran systems were examined using gravimetric analysis, modulated differential scanning calorimetry, and X-ray crystallography. It was shown that vitrification can be achieved isothermally without crystallization and that vitrification of trehalose solutions can be significantly accelerated by incorporating high-molecular-weight dextrans. Additionally, it was shown that, for the same water content, the glass transition temperature of the trehalose-dextran solution is significantly higher than that of the binary trehalose solution, making the glassy state achievable and storage feasible.

Introduction Preservation technologies currently in use (mainly, lyophilization and cryopreservation) depend on specialized, low-temperature and pressure equipment for processing or cryogenic temperatures for product storage. Nature, however, offers an alternative method for preservation by anhydrobiosis, where many organisms such as yeast, brine shrimp, tartigrade, and plant seeds can survive extreme draught conditions when they lose almost all of their water and resurrect when conditions are fair.1-3 The common behavior seen in all of these organisms is that they produce high concentrations of disaccharides (such as trehalose). It is already shown that disaccharides play a major role in dried preservation of seeds (for a review see ref 4). Trehalose is known to be effective in cryopreservation5 of mammalian cells, protection against dehydration-induced fusion of lipid membranes,6,7 and protein denaturation.8 One of the protection mechanisms is offered to be the “water-replacement hypothesis”,9 stabilizing the lipid and protein structures (for a complete review of the suggested protection mechanisms, see ref 10). The high glass transition temperature of trehalose * Corresponding author: Alptekin Aksan, Ph.D. Phone: (617) 371-4912. Fax: (617) 371-4950. E-mail: [email protected]. (1) Crowe, J. H.; Cooper, A. F. Sci. Am. 1971, 225, 30. (2) Sun, W. Q.; Irving, T. C.; Leopold, A. C. Physiol. Plant. 1994, 90, 621. (3) Crowe, J.; Hoekstra, F.; Crowe, L. Annu. Rev. Physiol. 1992, 54, 579. (4) Sun, W. Q. Ann. Bot. 1997, 79, 219. (5) Eroglu, A.; Russo, M. J.; Bieganski, R.; Fowler, A.; Cheley, S.; Bayley, H.; Toner, M. Nat. Biotechnol. 2000, 18, 163. (6) Crowe, L. M.; Crowe, J. H.; Rudolph, A.; Womersley, C.; Appel, L. Arch. Biochem. Biophys. 1985, 242, 240. (7) Chen, T.; Acker, J. P.; Eroglu, A.; Cheley, S.; Bayley, H.; Fowler, A.; Toner, M. L. Cryobiology 2001, 43, 168. (8) Carpenter, J. F.; Chang, B. S. Lyophilization of Protein Pharmaceuticals. In Biotechnology and Biopharmaceutical Manufacturing, Processing and Preservation; Avis, K. E., Wu, V. L., Eds.; Interpharm Press: Buffalo Grove, IL, 1996. (9) Crowe, L. M.; Mouradian, R.; Crowe, J. H.; Jackson, S. A.; Womersley, C. Biochim. Biophys. Acta 1984, 769, 141. (10) Crowe, J. H.; Crowe, L. M.; Oliver, A. E.; Tsvetkova, N.; Wolkers, W.; Tablin, F. Cryobiology 2001, 43, 89.

(when compared to other saccharides such as lactose, maltose, sucrose, glucose, and fructose) is also offered as another factor for why it forms an intra-/extracellular glass.3 The glassy (vitrified) state is a meta-stable supersaturated state characterized by very low molecular mobility (and, therefore, reduced diffusion), where there is an increase of approximately 9 orders of magnitude in molecular relaxation times. Decreased molecular mobility in the vitrified state ensures reduced chemical reaction (and, therefore, deterioration) rates. Mimicking nature, storage in the vitrified state (if it can be achieved and stabilized at room temperature, for example), therefore, appears to be a very economical method for the preservation of proteins, enzymes, and cells. Isothermal, isobaric vitrification does not require cryogenic temperatures; however, the long time periods required to reach the glassy state may render it impractical for preservation purposes. During isothermal drying, the free volume decreases while the viscosity and glass transition temperature of the solution increases.11 As a result of the desiccation-induced increase in viscosity and the corresponding decrease in water diffusivity, it is thought that vitrification is improbable in the experimental time scales considered to be feasible for the processing of biological materials. Mammalian cells survive dehydration down to a certain water content (∼50-60% of original water volume) without significant loss of viability but die upon removal of 1015% additional water. The tradeoff between the loss of cell viability during desiccation and the subsequent reduction in the molecular mobility due to reduced water content making storage feasible was the focus of this research. We suggest that high-molecular-weight polysaccharides (such as dextran) can be used to modify the isothermal desiccation and vitrification kinetics of certain sugars that have been known to be effective in protection against freezing and desiccation damage (such as trehalose, sucrose, or glucose). To explore this hypothesis, isothermal vitrification of a model trehalose-dextran (TD) system was explored. The suggested benefits of (11) Gotze, W. J. Phys.: Condens. Matter 1999, 11, A1.

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combining trehalose with high-molecular-weight sugars are as follows: (1) The glass transition temperature increases with molecular weight; therefore, it is expected that the addition of high-molecular-weight sugars will increase the glass transition temperature of the trehalose solution, making room-temperature storage feasible (dextran-sucrose solutions have higher glass transition temperatures following freeze-drying).12,13 It is shown that, for increased stability of the ingredients, the glass transition temperature should at least be 50K higher than the storage temperature.14 (2) The equilibrium water content of dextran is higher than that of trehalose and is known to increase with increasing molecular weight (in the range from 1500 to 20 000).13 It is, therefore, hypothesized here that the polydisaccharide mixture would carry more water at a given glass transition temperature. This will help lower the desiccation stress on cells during processing. (3) The proven efficiency of high-molecularweight dextran solutions in protecting certain enzymes15 and cell types16 against freezing damage is expected to increase the protective capacity of the drying solution. For example, the synergistic effect of disaccharidedextran mixtures is known to increase the storage stability of freeze-dried actin.17 Materials and Methods The promise of dried state preservation is based on the hypothesis that reaching a very low molecular mobility state enabling reduced chemical reaction and deterioration rates is the primary factor for the long-term stability of the preserved ingredients. If vitrification is the main mechanism of protection offered by a carbohydrate glass former, whether it is achieved through isothermal desiccation or lyophilization, the final state of the product determines its storage stability. Determination of the final state requires measurement of the primary indicator, the molecular mobility (by fluorescence recovery after photobleaching,18 nuclear magnetic resonance,19 or electron spin resonance),20 or of parameters such as the change in viscosity, density, mechanical strength, dielectric properties, or heat capacity, cp, during glass transition. In this research, we have focused on measuring the glass transition temperatures of isothermally dried specimens using modulated differential scanning calorimetry (MDSC). Differential scanning calorimetry (DSC) has been used extensively to determine the glass transition temperatures of various sugar solutions and polymers by examining the gross thermal response (heat flow in/out of the sample). In MDSC, the heat input to/from the sample can be deconvoluted into its reversible and irreversible components and the glass transition (a reversible process) can easily be distinguished from enthalpic relaxation (an irreversible process). Enthalpic relaxation is a function of the thermal history of the sample (not of the end state) and may create effects that can complicate the detection of glass transition during conventional DSC experimentation. All of the stock solutions to be used in the experiments were prepared gravimetrically on a microbalance using ultrapure water. High purity trehalose dihydrate (MW ) 378.3) was purchased from Pfanstiehl (Ferro Pfanstiehl Laboratories, Inc., (12) te Booy, M. P. W. M.; de Ruiter, R. A.; de Meere, A. L. J. Pharm. Res. 1992, 9, 109. (13) Imamura, K.; Fukushima, A.; Sakaura, K.; Sugita, T.; Sakiyama, T.; Nakanishi, K. J. Pharm. Sci. 2002, 91, 2175. (14) Hancock, B. C.; Shamblin, Z. L.; Zografi, G. Pharm. Res. 1995, 12, 799. (15) Ashwood-Smith, M. J.; Warby, C. Cryobiology 1972, 9, 137. (16) Pellerin-Mendes, C.; Million, L.; Marchand-Arvier, M.; Labrude, P.; Vigneron, C. Cryobiology 1997, 35, 173. (17) Allison, S. D.; Manning, M. C.; Randolph, T. W.; Middleton, K.; Davis, A.; Carpenter, J. F. J. Pharm. Sci. 2000, 89, 199. (18) Champion, D.; Hervet, D.; Blond, G.; Le Meste, M.; Simatos, D. J. Phys. Chem. B 1997, 101, 10674. (19) Moran, G. R.; Jeffrey, K. R. J. Chem. Phys. 1999, 110, 3472. (20) Biuitink, J.; Leprince, O.; Hemminga, M. A.; Hoekstra, F. A. Proc. Natl. Acad. Sci., U.S.A. 2000, 97, 2385.

Aksan and Toner Table 1. Stock Solutions Testeda solution number

solution

md

mT/mD

1 2 3 4 5 6 7

trehalose T-D (MWdextran ) 1.88 × 106) T-D (MWdextran ) 3.57 × 104) T-D (MWdextran ) 1.88 × 106) T-D (MWdextran ) 1.88 × 105) T-D (MWdextran ) 3.57 × 104) T-D (MWdextran ) 1.00 × 104)

0.3338 0.3161 0.3163 0.2983 0.2982 0.2987 0.2982

N/A 3.812 3.800 1.271 1.265 1.267 1.265

a m : dry matter mass/total mass. m /m : trehalose mass/ d T D dextran mass.

Waukegan, IL). Industrial and research grade dextrans at various molecular weights (average MW ) 1.0 × 104, 1.5 × 104, 3.57 × 104, 1.44 × 105, 1.88 × 105, 1.88 × 106 according to the manufacturer’s analysis) were purchased from Sigma (Sigma-Aldrich Corp., St. Louis, MO). Prior to preparation, all of the chemicals were kept in an 85 °C atmospheric oven for 24 h. The state of trehalose was determined using a differential scanning calorimeter (model 2920, TA Instruments, New Castle, DE) to be anhydrous (absence of dehydration endotherm at 100 °C and R-trehalose melting endotherm at 120.9 °C21 showed that trehalose was anhydrous). For detailed information on the structure of anhydrous trehalose, see Willart et al.22 and the references therein. The water content of the baked trehalose powder, measured by a moisture analyzer (HR73-P, Mettler Toledo, Columbus, OH) was less than mw ) 0.001 (mw ) mwater/ mdrymatter). In the moisture analyzer, the sample was heated to 175 °C while its mass was continuously monitored. The experiment was terminated when no more change in the sample mass was observed (initial sample mass was approximately 10 g and the sensitivity of the balance was 0.1 mg). The moisture contents of the dextran powders were below mw ) 0.001. Binary and ternary stock solutions from anhydrous trehalose and dried dextran powder (at mainly two different mass ratios mtrehalose/ mdextran ) 1.3, 3.9) were prepared inside a glovebox pressurized with nitrogen gas (relative humidity, RH < 4%). The water contents of all of the stock solutions prepared were measured gravimetrically and using the moisture analyzer (the difference between the results from these two methods was less than 0.3% for all of the solutions tested). In Table 1, the experimental groups are presented. To determine the isothermal desiccation kinetics, a high sensitivity (∼1 µg) microbalance (Cahn C-33, Thermo Orion Co., Beverly, MA) placed in a nitrogen pressurized glovebox kept at 0% RH, 22.4 °C, was connected to a personal computer through an RS-232 interface and the water loss from 10 µL of trehalose and dextran solutions [initially containing 30% (w/w) sugar] were measured as a function of the drying time. The data were collected at 1 Hz, for up to 1.5 million s under constant humidity ((0.25% RH) and temperature ((0.5 °C) conditions. Before drying, all of the stock solutions were mixed in a vortex mixer and filtered (filter pore size was 0.45 µm). A total of 10 µL of stock solutions were transferred to aluminum DSC pans. It was ensured that the sample solution wetted the botton of the aluminum pan completely. The internal diameter of the aluminum dishes was 5 mm, and the initial thickness of the sample solution was approximately 500 µm. Triplicate samples from each solution were also transferred on microscope coverslips for microscopic analysis and quartz crystal microbalance analysis (results not reported here). These samples were subjected to the same drying conditions as the aluminum DSC pans. The aluminum pans, the caps, and the samples before and after drying were weighed using a high precision microbalance (MT5 Mettler Toledo, Columbus, OH). The sensitivity of the balance was 0.1 µg. The uncapped pans were then desiccated isothermally either at 24.4 or 37 °C (additional experiments were performed also at 4, 55, 65, 75, 85, and 95 °C) and 0% RH by storing inside sealed boxes (over excess amounts of CaSO4) over an extended period of time (td < 700 h). The humidity and temperature inside the (21) Sussich, F.; Cesaro, A. J. Therm. Anal. Calorim. 2000, 62, 757. (22) Willart, J. F.; De Gusseme, A.; Hemon, S.; Descamps, M.; Leveiller, F.; Rameau, A. J. Phys. Chem. B 2002, 106, 3365.

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Figure 1. Desiccation regimes of 30% trehalose and dextran solutions. (The area bounded by the dashed line is magnified in the inset.) drying boxes were continuously monitored using a precision thermo-hygrometer. Fluctuations in the temperature and humidity during drying were less than 0.5 °C and 1%, respectively. At certain time intervals, selected sample pans were taken out of the dryboxes, weighed, and hermetically sealed. The glass transition temperature of the sample was determined using MDSC. In a typical isobaric scan (constant temperature change heating or cooling), glass transition took place over a temperature range. The midpoint of the baseline shift in the heat capacity signal was taken as the glass transition temperature, Tg, of the particular specimen tested. The calorimeter was calibrated for temperature using the known melting points of zinc, indium, and tin and for heat capacity measurement using the literature values of sapphire. The calorimeter was operated in temperature modulation mode, at a temperature modulation amplitude of 0.5 °C and a modulation period of 60 s. Heat flow to/from the hermetically sealed sample pan (normalized with respect to a hermetically sealed reference pan whose weight matched the sample pan within 0.1 mg) as well as the heat capacity of the sample was recorded at 10 Hz as a function of temperature. Both sides of the calorimeter heating cell were purged with ultrapure nitrogen gas at a flowrate of 25 mL/min. Starting from room temperature, the temperature of the specimen was lowered to -60 °C at the maximum rate allowed by the calorimeter and kept constant for 10 min. The sample was then heated at a rate of 10 °C/min to an end point temperature between 70 and 250 °C. The samples heated to lower end point temperatures were later used for the confirmation of the sample water content by opening the sealed pans, baking at 95 °C for 48 h, and recording the weight loss. The samples heated to high end point temperatures were used to confirm cold crystallization and crystal melting (examined in detail in Results). To examine the effects of sample-drying surface interactions, selected aluminum sample pans were coated with a Krytox (DuPont, Wilmington, DE) dry film and the drying rates and the final structural state of the specimens were compared to those of samples dried on aluminum surfaces. To test for crystallinity, powder X-ray crystallography experiments with selected dried samples were performed. For these experiments, 10 µL of trehalose and T-D solutions were pipetted on regular (wetting contact) and Krytox-coated (nonwetting)

microscope slides and dried for 168, 1056, or 2160 h at 24.4 °C in a 0% RH environment. Sealed samples were then examined using a Bruker Smart CCD diffractometer system (Siemens AG, Germany).

Results Desiccation Kinetics. Observed by gravimetric experimentation performed for up to 500 h, the isothermal desiccation behavior of dextran can be divided into three regimes: (a) an initial rapid, constant drying rate regime; (b) a slowed desiccation regime due to the formation of a rubbery, thin skin (corresponding to point 1 in Figure 1) on the surface of the droplet lowering the evaporation rate, where the core of the droplet was still liquid; and (c) a homogeneous gel phase, where the thickness of the skin increased and finally extended through the thickness of the droplet (point 2 in Figure 1). It is known that during drying due to increased viscosity in the sample (and, therefore, reduced diffusivity), the surface of the samples dries faster than the core and a sticky, glassy crust forms on the surface reducing the surface water evaporation rate.23 Initial skin formation decelerated desiccation slightly. However, with the timedependent increase in the thickness of the skin (gellification), the desiccation rate decreased significantly. The trehalose specimen went through the same desiccation regimes as the dextran droplet even though the effects of skin formation and uniform gellification on the evaporation rate were more significant. Additionally, with further drying a fourth desiccation regime for trehalose was discovered to be starting at a time point corresponding to mw ) 0.105, where there was an abrupt increase in the desiccation rate (point 3 in Figure 1). The reason for the existence of a fourth desiccation regime for trehalose, starting at this particular water content, is examined in (23) Romdhane, I. H.; Price, P. E.; Miller, C. A.; Benson, P. T.; Wang, S. Ind. Eng. Chem. Res. 2001, 40, 3065.

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Figure 2. Typical MDSC scan for the trehalose solution (only the heating data are shown).

the next section. After homogeneous gellification, the water contents of the dextran samples were always lower than those for trehalose at the same time point. The desiccation responses of T-D mixtures fell between the desiccation curves presented for trehalose and dextran and are not shown in Figure 1 for clarity. Vitrification Kinetics. To quantify the glass transition temperature of the dried specimens, DSC experiments were performed at a scanning rate of 10 °C/min as explained in Materials and Methods. The samples tested immediately (without drying) showed two broad endothermic peaks at approximately 0 and 100 °C. These peaks corresponded to the melting and evaporation of the free water in the sample, respectively. Trehalose samples dried for a minimum of 2 h (td ) 2 h) in a 0% RH environment lacked these endotherms. In a typical DCS experiment with dried samples (td > 2 h), with increasing temperature, initially a flat baseline followed by a shift in the heat flow to the specimen (accompanied by a similar shift in the specific heat) was observed (Figure 2). The shift in the baseline is the characteristic sign of the glass transition.24 The enthalpic overshoot located at the end temperature of the glass transition, Tge, is characteristic of glass-forming sugars. The glass transition temperature, Tg, was calculated at the midpoint of the tangential lines drawn to the baselines before, Tgs, and after, Tge, glass transition. With further heating of the specimen, cold crystallization (an exothermic peak corresponding to the crystallization of trehalose β from its rubbery phase)21 followed by an endothermic peak corresponding to the melting of trehalose β crystals21 was observed. Note that, in Figure 2, the magnitudes of the cold crystallization exotherm and crystal melting endotherm are not equal. This is mainly due to the sensitivity of cold crystallization to the scan rate. At very low scanning rates, because all of the molecules have enough time to form crystals and then melt simultaneously, the magnitudes would, theoretically, be equal.21 The melting temperature was calculated at the endothermic maxima of the heat capacity signal. For the trehalose solutions tested, the melting temperature, Tc, was 207.6 ( 1.3 °C. This value is in good agreement with the literature (for an extensive review, see Sussich and Cesaro).21 Compartmentalization of the sugars was not detected (in all of the DSC scans, a single, clear glass transition was observed). This showed the compatibility and miscibility of the sugars used. (24) Blair, H. E. ASTM Spec. Tech. Pub. 1994, 1249, 50.

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Trehalose Solutions. Three different vitrification regimes in terms of the change in the glass transition temperature were observed. In the first regime, the glass transition temperature showed a logarithmic dependence (R2 ∼ 0.92) on the drying time, td. However, the samples have not reached the glassy state (Tg < Td) with isothermal (Td ) 24.4 °C), atmospheric drying up to 300 h at 0% RH (open circles in Figure 3). With increased proximity to the glassy state, the translational mobility of the water molecules was progressively hindered, slowing down evaporation. Beyond 300 h of drying, however, the glass transition temperature of the trehalose samples increased very rapidly and reached approximately 91.4 °C after 450 h (second drying regime). With further drying, a third regime characterized by a slow increase in the glass transition temperature was observed. It is important to note that the inflection point of the transition between the first and the second drying regimes corresponded to mw ) 0.105. Note that a similar transition behavior was observed in the desiccation rate as well, which corresponded to the same water content. A water ratio of 0.105 is equal to the water content of trehalose dihydrate. The theoretical value for the trehalose dihydrate water content is 0.105 16, and the experimentally measured value is 0.104 (experiments are not reported here). Experiments performed with the trehalose solutions dried isothermally at 37 °C, 0% RH, showed a similar transition (from the first drying regime to the second) at a location corresponding to mw ) 0.105 as well. This showed that the transition inflection point was independent of the isothermal drying temperature. The maximum plateau glass transition temperature of the isothermally dried trehalose specimens measured after 700 h (Tg ) 97.2 °C) were still below the glass transition temperatures of the dihydrate (Tg ) 112.6 ( 1.4 °C) and amorphous (Tg ) 118.2 ( 0.7 °C) trehalose. This was attributed to the residual water (mw < 0.02) trapped in the specimen. No obvious correlation between ∆Cp/Cp measured at glass transition and Tg or td was observed. ∆Cp/Cp remained fairly constant around 0.4 for all of the samples tested, indicating that the samples were homogeneous (within the detection capabilities of MDSC). In Figure 4, thermograms from two trehalose solutions dried for td ∼ 450 h (Td ) 24.4 °C, Tg ) 38.08 °C) and td ∼ 700 h (Td ) 24.4 °C, Tg ) 95.12 °C) are compared to that for anhydrous trehalose R (td ) 24 h, Td ) 85 °C, Tg ) 117.32 °C). Note that trehalose R and β are the two crystalline forms of anhydrous trehalose (a third crystalline form is trehalose dihydrate). The thermograms of the trehalose solutions (Figure 4) lacked the peaks corresponding to the dehydration of trehalose dihydrate (∼100 °C), melting of trehalose R crystals (which, for example, exists in the thermogram for anhydrous trehalose), and melting of trehalose γ crystals (130-145 °C). Trehalose γ is a combination of trehalose dihydrate and trehalose β crystals.25 The thermograms for dried trehalose solutions also showed cold crystallization followed by the trehalose β melting endotherm further supplying the calorimetric evidence that the dried trehalose solutions did not have a crystalline structure. T-D Solutions. The T-D solutions tested showed similar DSC scans: a flat baseline interrupted by a step change (indicating the glass transition), the cold crystallization exotherm, and the trehalose melting endotherm. Compared to the trehalose solutions, the temperature range of glass transition (Tge - Tgs) was larger. The (25) Sussich, F.; Princivalle, F.; Cesaro, A. Carbohydr. Res. 1999, 322, 113.

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Figure 3. Glass transition temperature, Tg, as a function of drying time, td. Dashed line: drying temperature, Td ) 24.4 °C.

Figure 4. DSC thermograms of high Tg trehalose specimens.

vitrification curve of T-D solutions also consisted of three regimes. However, the vitrification rates in the first regime were significantly higher than those of the trehalose solutions such that the high-mass-ratio T-D solutions (mtrehalose/mdextran ∼ 3.9 group shown by crosses in Figure 3) were glassy after only 11 h of drying. It took approximately 60 h for the low-mass-ratio T-D solutions (mtrehalose/mdextran ∼ 1.3) to reach the glassy state (as shown by the hollow squares and triangles in Figure 3). The transition from the first to second vitrification regimes (even though the inflection point was less and less pronounced with decreasing trehalose content in the T-D mixture) corresponded to mw values proportional to the mass ratio of trehalose in the T-D solution. For the same drying time, the glass transition temperature of the T-D solution increased (∼5K) when the molecular weight of the dextran used in the T-D solution was increased (for example, from 3.57 × 104 to 1.88 × 106). However, the increase in the glass transition temperature was more significant when the mass ratio of dextran to trehalose in the solution was increased (∼20-25K). In Figure 5, the state diagram of the T-D system is shown. All of the solutions experimented with have shown increased glass transition temperature with reduced water content. With increasing dextran-to-trehalose mass ratio in the system, the glass transition temperature of the

solution increased significantly. Here again, the effect of higher-molecular-weight dextrans was not significant. Note that for trehalose solutions, the Gordon-Taylor equation (see Chen et al. for details) fitted the glass transition curve with a k of 5.4 (this is very close to the k ) 5.2 value determined using all of the available data in the literature).26 The same constant was calculated as 5.7 for dextran. In Figure 6, X-ray crystallography results are presented. In the positive controls (trehalose dihydrate specimens), the presence of crystalline structures was confirmed (the white specks). None of the dried trehalose or T-D samples showed a crystalline structure (after 2160 h of isobaric, isothermal drying at 0% RH, 24.4 °C), confirming our calorimetric findings. The samples dried on aluminum pans (except highmolecular-weight T-D solutions) and the microscope coverslips had developed extensive surface cracks (Figure 7). It was observed that the cracking patterns differed according to the composition of the test sample. Trehalose specimens had very random micro- and macroscale cracks (Figure 7A). Dried trehalose solutions prepared with lowermolecular-weight dextrans (MW ) 1.00 × 104, 3.75 × 104) had uniformly distributed cracks large in size but few in number (Figure 7B,C). Trehalose solutions with highmolecular-weight dextrans (MW ) 1.88 × 105, 1.88 × 106) showed no cracks on their surfaces (Figure 7D,E). Both the trehalose and T-D drops had significantly different peripheral regions that are crossed by (with increasing distance from the center) initially radially and then tangentially oriented cracks. These patterns are very similar to those reported in the literature for the silicabased sol-gel drops.29 Irrespective of the solution composition, the samples dried in Krytox coated pans and coverslips did not have cracking. Cracking is thought to be caused by the surface stresses and secondary flows within the sample created by the peripheral pinning of a drying drop on a surface it wets.27,28 Note that the droplet(26) Chen, T.; Fowler, A.; Toner, M. Cryobiology 2000, 40, 277. (27) Deegan, R. D.; Bakajin, O.; Dupont, T. F.; Huber, G.; Nagel, S. R.; Witten, T. A. Nature 1997, 389, 827. (28) Deegan, R. D.; Bakajin, O.; Dupont, T. F.; Huber, G.; Nagel, S. R.; Witten, T. A. Phys. Rev. E 2000, 62, 756. (29) Caddock, B. D.; Hull, D. J. Mater. Sci. 2002, 37, 825.

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Figure 5. State diagram for trehalose and T-D solutions: glass transition temperature, Tg, as a function of the water mass ratio, mw. Dashed lines 1 and 2, curve fit to data from this research; solid line 3, trehalose data from ref 38; solid gray line 4, dextran data from ref 39.

Figure 6. X-ray crystallography of dried samples with 2160 h of isobaric, isothermal drying at 0% RH and 24.4 °C.

drying surface interactions also determine the drying rate30 such that the specimens dried in Krytox coated pans dried slower irrespective of the sample composition (as a result of decreased surface area and absence of secondary flows within the specimen). Discussion It was shown that, with isothermal, isobaric drying at 0% RH, it was possible to reach the glassy state for a trehalose solution and a T-D system without crystallization. With the addition of high-molecular-weight sugars, vitrification rates of isothermally dried carbohydrate solutions were significantly accelerated as a function of the dextran mass ratio in the sample. For the same water content, the resultant glass transition temperature of the T-D system was higher than binary trehalose solutions. With increasing the molecular weight of dextran in the solution, it was also possible to achieve (if its mass ratio (30) Bourges-Monnier, C.; Shanahan, M. E. R. Langmuir 1995, 11, 2820.

to trehalose was constant) a higher Tg at the same water content. The crack formation on the surface of the dried droplets was mainly caused by the droplet-drying surface interactions. Cracks could be eliminated by increasing the molecular weight of dextran in the solution and modifying the drying surface. Vitrification Kinetics. Increased Tg with poly(acrylic acid)-dextran32 and sucrose-dextran systems12,13,33 is reported in the literature. Here, the same effect of dextran is shown for the trehalose systems with additional advantages during isothermal drying conditions examined. Imamura et al. report an increased Tg difference as a function of dextran molecular weight (for MWdextran ) 1500, 6000, 20 000), with a constant dextran-to-sucrose mass ratio.13 Similar to their observations, for high mtrehalose/mdextran ratio solutions at low water contents, the glass transition temperatures of the T-D (MWdextran ) 37 500) and T-D (MWdextran ) 1.88 × 106) solutions were observed to be significantly different (∼20 °C). However, the same difference was not observed for the low mtrehalose/ mdextran ratio solutions. In dilute solutions, even if the free volume remains the same, due to increased entanglement, higher-molecular-weight dextrans are expected to present a bigger obstacle for water diffusion because this is shown to be the case by quasi-elastic light scattering spectroscopy for the reduced diffusion of 67-nm latex spheres in dextran solutions with increased molecular weight.34 However, below a certain free volume, as a result of steric hindrance, the sheer mass of the dextran molecules surrounding the water molecule (rather than their entanglement) appears to be the dominant factor. On the basis of the same point of view, the low sensitivity of the sample vitrification rate to the dextran molecular weight is not surprising. The initial water content of the T-D stock solutions was less than (though not significantly) that of the (31) Gallo, A.; Buera, M. P.; Herrera, M. L. J. Food Sci. 2002, 67, 1331. (32) Cascone, M. G.; Polacco, G.; Lazzeri, L.; Barbani, N. J. Appl. Polym. Sci. 1997, 66, 2089. (33) Shamblin, S. L.; Taylor, L. S.; Zografi, G. J. Pharm. Sci. 1998, 87, 694. (34) Phillies, G. D. J.; Quinlan, C. A. Macromolecules 1992, 25, 3110.

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Figure 7. Isothermal drying crack formation mtrehalose/mdextran ∼ 1.3.

trehalose stock solutions (the difference was Tg,u). As a result of excess water influx from the crystallizing region 1 to region 3, the glass transition temperature in region 3 drops significantly (Tg,3 , Tg,u). In the same figure (Figure 8) the schematic DSC thermograms corresponding to these two samples are given. The uniform trehalose glass undergoes a single glass transition (Tg,u) while the thermogram corresponding to the compartmentalized solution has (at least) two located at temperatures Tg,3 and Tg,2. In the limiting case, the calorimetric effect of an infinite number of compartments within a sample (which corresponds to a water content gradient) would be reflected as an increase in the temperature range (Tge - Tgs) over (36) Sun, W. Q.; Davidson, P. Biochim. Biophys. Acta 1998, 1425, 235.

Aksan and Toner

which the glass transition takes place. In our experiments, we have not seen any of these effects. The absolute magnitude of the step change in the heat capacity signal during glass transition (∆Cp) is directly proportional to the size of its corresponding subregion. The endotherm located at ∼100 °C on the thermogram for the compartmentalized solution corresponds to the dehyration of the trehalose dihydrate crystal in region 1 (evaporation of the two water molecules in the dihydrate crystal). There is also another peak located at ∼130-145 °C corresponding to the melting of the anhydrous trehalose crystals which form by cold crystallization following the loss of the bound water (not shown in Figure 8). In a dominantly crystalline sample, the magnitude of the ∆Cp/ Cp ratio decreases with drying time because the volume of the glassy subregion decreases in time. This theory is valid under the assumption that the scanning rate during DSC analysis is high enough that water diffusion between subregions at different states is negligible. In the case of locally evaporating water, which is more likely to be seen in drying of very thin films, the DSC thermogram is expected to have a higher Tg and dihydrate dehydration and melting endotherms. According to NMR data presented by Gallo et al.,31 16% of the 72.5% (w/w) supersaturated trehalose solution crystallizes after only 400 min at 25 °C. Crystallization of this magnitude is expected to produce significant dehydration and melting endotherms, which clearly are missing in our samples. The X-ray crystallography experiments and microscopic examination confirmed the MDSC results that none of the dried trehalose or T-D samples tested had crystalline structure (Figure 6). Librizzi et al.37 report three different drying regimes for isothermal drying of 0.5 mL of trehalose solutions on the basis of Fourier transform infrared spectroscopy measurements. They attribute the transition to the third drying regime to microcrystallization within the droplet. The increase they record in turbidity measurements supports their claims. In isothermal high-temperature drying experiments, we have not been able to determine the third regime (which is thought to be caused by crystallization) up to 48 h of drying at 85 °C. However, during isothermal drying experiments at room temperature, the change in vitrification kinetics as a function of time was evident (Figure 3). In any case, MDSC analysis has not indicated any significant crystallization in the macroscale. The effect of microcrystallization on the ingredients of the glass is yet to be examined. Note that the efficacy of the preservation solution is not only a function of its dried state but also of chemical and biological factors that need to be engineered. To examine the feasibility of adapting an isothermal vitrification method for preservation of living systems, future studies will involve measurement of drying kinetics and vitrification of carbohydrate solutions in the presence of cells. Conclusion To achieve long-term storage stability, molecular mobility should be significantly reduced within and outside a mammalian cell. This requires uploading the cells with as well as desiccating them in a medium that contains glass-forming carbohydrates. Cells survive dehydration down to a certain water content (∼50-60% of the original water volume) without significant loss of viability but die upon removal of 10-15% additional water. The tradeoff (37) Librizzi, F.; Vitrano, E.; Cordone, L. Biophys. J. 1999, 76, 2727. (38) Miller, D. P.; de Pablo, J. J.; Corti, H. Pharm. Res. 1997, 14, 578. (39) Cojazzi, G.; Pizzoli, M. Macromol. Chem. Phys. 1999, 200, 2356.

Trehalose-Dextran Solutions

between the loss of viability during desiccation and the subsequent reduction in molecular mobility due to reduced water content making storage feasible (especially in the presence of glass formers such as trehalose) presents a dilemma beyond which the key to desiccated state preservation lies. In this research, desiccation and vitrification kinetics of model trehalose and T-D systems were examined using gravimetric analysis, MDSC, and X-ray crystallography. Three different desiccation regimes for dextran and four desiccation regimes for trehalose have been established. It was shown for these systems that vitrification can be achieved isothermally without crystallization and vitrification of trehalose solutions can be significantly accelerated by incorporating high-molecular-weight dextrans. Additionally, it was shown that, for the same water

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content, the glass transition temperature of the T-D solution is significantly higher than that of the binary trehalose solution, making dried-state storage more feasible. Acknowledgment. We thank the director of the X-ray Crystallographic Laboratory of Department of Chemistry at Harvard University, Dr. Richard J. Staples, for performing the X-ray crystallography analysis. We also thank the anonymous reviewers for their constructive suggestions and comments. This research is funded by a National Institutes of Health (DK46270) grant and Defense Advanced Research Projects Agency/Naval Research Projects (N00173-01-1 G011) grant. LA0355186