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Analysis of Desiccation and Vitrification Characteristics of Carbohydrate Films by Shear-Wave Resonators Alptekin Aksan,*,† Scott C. Morris,‡ and Mehmet Toner† Center for Engineering in Medicine and Surgical Services, Massachusetts General Hospital, Harvard Medical School, and Shriners Hospital for Children, Boston Massachusetts 02114, and Department of Aerospace and Mechanical Engineering, University of Notre Dame, Notre Dame, Indiana 56556 Received September 7, 2004. In Final Form: December 27, 2004 Desiccated state preservation of mammalian cells and tissues in the presence of carbohydrates has started to show promise in the last two decades. Certain carbohydrates play a major role in preservation by reducing molecular mobility in the desiccated state. In this communication, the feasibility of utilizing shear-wave resonators to collect real-time molecular mobility information during desiccation and vitrification of carbohydrate based thin films was demonstrated. Simultaneous quartz crystal microbalance experimentation and optical imaging were utilized to determine the conditions for thin film formation and the vitrification kinetics of certain carbohydrate solutions of biological importance. Using the technique presented here, it was possible to gain insight into the vitrification characteristics of carbohydrate solutions establishing the basics for future research with quantitative analysis of film properties and experimentation with live mammalian cells.
Introduction 1
2
Certain organisms (such as escherichia coli, yeast, and nematodes3) are known to synthesize carbohydrates upon exposure to extreme temperatures and desiccation, a function that is associated with their survival.4 Discovery of a carbohydrate-based vitrified state in the cytoplasm of desiccated plant seeds,5 Artemia cysts,6 and fungal spores supplies additional evidence on the role of sugars in “suspended animation”.7 Mimicking nature, over the last two decades, researchers have been trying to utilize carbohydrates to preserve mammalian cells in the frozen (cryopreservation) or desiccated state.8-15 Through these efforts it was established that for successful stabilization in the preserved state, carbohydrates (glucose, sucrose, raffinose, and stachyose in plants, and trehalose in microorganisms and animals) should be present on both sides of the cell membrane.16 In many respects, the † Massachusetts General Hospital, Harvard Medical School, and Shriners Hospital for Children. ‡ University of Notre Dame.
(1) Kandror, O.; DeLeon, A.; Goldberg, A. L. Proc. Natl. Acad. Sci. U.S.A. 2002, 99, 9727. (2) Attfield, P. V. FEBS Lett. 1987, 225, 259. (3) Madin, K. A. C.; Crowe, J. H. J. Exp. Zool. 1975, 193, 335. (4) Crowe, J. H.; Hoekstra, F. A.; Crowe, L. M. Annu. Revi. Physiol. 1992, 54, 579. (5) Williams, R. J.; Leopold, A. C. Plant Physiol. 1989, 89, 977. (6) Clegg, J. S.; Seitz, P.; Seitz, W.; Hazlewood, C. F. Cryobiology 1982, 19, 306. (7) Crowe, J. H.; Cooper, A. F. Sci. Am. 1971, 225, 30. (8) Miller, D. P.; de Pablo, J. J.; Corti, H. Pharm. Res. 1997, 14, 578. (9) Conrad, P. B.; Miller, C. A.; Cielenski, P. R.; de Pablo, J. J. Cryobiology 2000, 41, 17. (10) Miller, D. P.; Anderson, R. E.; de Pablo, J. J. Pharm. Res. 1998, 15, 1215. (11) Crowe, J. H.; Crowe, L. M.; Chapman, D. Science 1984, 223, 701. (12) Crowe, J. H.; Crowe, L. M.; Carpenter, J. F.; Rudolph, A. S.; Wistrom, C. A.; Spargo, B. J.; Anchordoguy, T. J. Biochim. Biophys. Acta 1988, 947, 367. (13) Crowe, J. H.; Crowe, L. M.; Odell, S. J. Cryobiology 1980, 17, 622. (14) Crowe, J. H.; Crowe, L. M.; Oliver, A. E.; Tsvetkova, N.; Wolkers, W.; Tablin, F. Cryobiology 2001, 43, 89. (15) Crowe, J. H.; Leslie, S. B.; Crowe, L. M. Cryobiology 1994, 31, 355.
disaccharide trehalose has been shown to be superior in its protective capacity against freezing and desiccation stresses. Therefore, it is in the center of many research efforts.8-15 The mechanism of stabilization offered by trehalose is hypothesized to be related to its high affinity to interact with the other solutes and surfaces either by replacing their vicinal water (the “water replacement hypothesis”17) or by enforcing partitioning (the “preferential exclusion hypothesis”18,19) during desiccation. Recently, a mechanistic explanation, based on molecular mobility measurements, was offered to explain the superior protective capacity of trehalose based on the higher rigidity of the matrix (when compared to those formed by the other disaccharides such as sucrose and maltose) it forms at low water concentrations.20 The high glass transition temperature of trehalose is another important factor for desiccated state preservation,21 where key to success is stopping molecular mobility, ideally by reaching a glassy state in and around the cell. For preservation in the desiccated state, carbohydrates (such as trehalose, which is normally membrane impermeable) should be artificially introduced into the cells (see Acker et al.16 for a recent review of available methods) and intra-/extracellular water should be removed in order to reduce molecular mobility. Almost all of the osmotically active water in a mammalian cell can be removed without any permanent damage. The osmotically inactive (vicinal)water on the other hand, is tightly associated with the ions, proteins, membranes, organelles, and macromolecules in the cytoplasm and upon removal may cause irreversible denaturation of these structures. Therefore, there is a minimum amount of water (which is cell type (16) Acker, J. P.; Chen, T.; Fowler, A.; Toner, M. Engineering Desiccation Tolerance in Mammalian Cells: Tools and Techniques. In Life in the Frozen State; CRC Press: Boca Raton, FL, 2004; p 563. (17) Crowe, J. H.; Crowe, L. M. Cryobiology 1982, 19, 670. (18) Xie, G.; Timasheff, S. N. Protein Sci. 1997, 6, 211. (19) Arakawa, T.; Timasheff, S. N. Biochemistry 1982, 21, 6536. (20) Migliardo, F.; Magazu, S.; Migliardo, M. J. Mol. Liquids 2004, 110, 11. (21) Aksan, A.; Toner, M. Langmuir 2004, 20, 5521.
10.1021/la047760y CCC: $30.25 © 2005 American Chemical Society Published on Web 03/01/2005
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dependent, in addition to other factors) below which, irrespective of the exposure time, the cell loses viability. The long processing times required for desiccation to a low enough water content to warrant reduced mobility also exposes cells to increasingly high osmotic stresses and thus may be fatal. One way of decreasing the desiccation time required is by reaching the glassy state at higher water content, with the additional benefit of ensuring that the intracellular water content does not fall below the critical vicinal water limit. In a recent communication, the feasibility of utilizing high molecular weight carbohydrates to accelerate the vitrification kinetics of trehalose during isothermal desiccation and to increase the water content at glass transition has been shown.21 To further increase the desiccation rate of the carbohydrate solutions (with the ultimate goal of minimizing the exposure time of the cells to high osmotic stresses), an alternative method is to increase the surface area-to-volume ratio of the solution by forming thin films. However, formation of a film at a uniform thickness by isothermal desiccation is not trivial since the drying characteristics of glass-forming carbohydrate solutions are very much dependent on their initial concentrations (the environmental conditions being constant). For very dilute solutions, the drying rate is limited by the diffusivity of water in the surrounding air (it is therefore a function of the air temperature and the relative humidity). With increased concentration however, drying rate becomes limited by the diffusivity of water within a droplet rather than that in the surrounding air. During drying, closer to the surface, the concentration gradients get extremely steep, and on the surface of the droplet a glassy skin is formed. The formation of the skin can easily be detected by physically touching the surface. It is hypothesized that the thickness of the skin is approximately 100 nm22 and does not present a high resistance to evaporation.23 The solid skin layer, however, offers mechanical resistance and therefore indirectly affects evaporation by delaying (and sometimes completely stopping) the movement of the droplet contact line. Due to the skin formation, even very small droplets of glass-forming solutions may dry nonuniformly, retaining a three-dimensional, nonuniform shape, rather than forming a film. Therefore, in the first part of the research presented here, the conditions required for the formation of thin films by isothermal desiccation were explored. In the second part of the research, desiccation experiments with carbohydrate films were performed to determine their vitrification characteristics. Determination of the vitrification kinetics of thin films of glass-forming carbohydrate solutions is crucial (a) to establish the differences of the carbohydrate solutions and the glasses they form in terms of their physical and mechanical properties, (b) to be able determine the state of the carbohydrate solution (containing cells to be preserved) during desiccation in real time, and (c) to reach the optimum water content in terms of cellular viability and molecular mobility. The tradeoff between the reduced cellular viability during desiccation and the subsequent reduction in molecular mobility making storage feasible presents a dilemma beyond which the key to desiccated state preservation lies. In this communication therefore, we examine the feasibility of utilizing quartz crystal resonators, first, to explore the vitrification kinetics of thin films of biologically important carbohydrate solutions in order to establish their differences and, second, to develop a useful tool for the desiccated state preservation research currently underway in our laboratory. (22) de Gennes, P. G. Eur. Phys. J. E 2002, 7, 31. (23) Pauchard, L.; Allain, C. Europhys. Lett. 2003, 62, 897.
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Theory Quartz crystal microbalance (QCM) response to a mass or a liquid layer deposited on its surface is quantified extensively in the literature24-27 for industrial as well as biomedical research applications. The decrease in the output frequency, ∆Fm, of the QCM due to the presence of a very thin, uniform thickness, mass layer deposited on its surface is given by the Sauerbrey equation as28
∆Fm ) -
2fo2 1/2
(Fqµq)
∆m Ao
(1a)
where ∆m, fo, Ao, Fq, and µq are the incremental mass deposited, the resonance frequency, the active (contact) surface area, the mass density (Fq ) 2648 kg/m3), and the shear modulus (µq ) 2.947 × 1010 kg/m‚s2) of the goldcoated AT-cut crystal, respectively. If, on the other hand, the deposited layer is a liquid, the output frequency decreases as a function of the mass density, Fl, and the absolute viscosity, ηl, of the liquid. The change in the frequency, ∆Fl, is then given by the Kanazawa-Gordon model as29
∆Fl ) -
fo3/2 π1/2(Fqµq)1/2
(Flηl)1/2
(1b)
For a drying droplet deposited at the center of the quartz crystal, at any point in time, the overall frequency shift, ∆F, can be determined by the simultaneous contributions from the mass and liquid loading and is given by30
∆F ) ∆Fm +
Ao ∆Fl A
(2)
where A is the overall surface area of the QCM crystal. In addition to the change in frequency, liquid loading also causes a change in the series resistance, ∆R, of the Butterworth van Dyke equivalent electrical circuit given by31
∆R )
4Luπ1/2fo3/2 (Flηl)1/2 (Fqµq)1/2
(3)
where Lu is the inductance for the unperturbed (dry) resonator. Materials and Methods The desiccation experiments were performed using a quartz crystal microbalance (QCM100, Stanford Research Systems Inc., Sunnyvale, CA) operating at 5 MHz placed in an environmental chamber, where the relative humidity (RH) could be controlled within 1% of the desired value by initially purging with ultradry nitrogen gas (∼0% RH) and moist air (∼90% RH) mixed at different proportions. To ensure diffusive drying during the experiments, gas circulation was stopped. The frequency and resistance outputs from the QCM were monitored at 1 Hz using a frequency counter (53131A, Agilent Technologies Inc., Palo (24) Lu, C.-S.; Lewis, O. J. Appl. Phys. 1972, 43, 4385. (25) Rodahl, M.; Kasemo, B. Sens. Actuators, A 1996, 54, 448. (26) Martin, S. J.; Frye, G. C.; Wessendorf, K. O. Sens. Actuators, A 1994, 44, 209. (27) Glassford, A. P. M. J. Vac. Sci. Technol. 1978, 15, 1836. (28) Sauerbrey, G. Z. Phys. 1959, 155, 206. (29) Kanazawa, K. K.; Gordon, J. G. Anal. Chim. Acta 1985, 175, 99. (30) Lin, Z.; Hill, R. M.; Davis, H. T.; Ward, M. D. Langmuir 1994, 10, 4060. (31) Henderson, J. Electronic Devices. Concepts and Applications; Prentice Hall: Englewood Cliffs, NJ, 1991.
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Figure 1. QCM response to a drying water droplet (Vi ) 0.2 µL, T ) 24.5 °C, φ ) 0% RH, polished crystal). Note: In the graph presented above, ∆R values were multiplied by 4 for visual clarity. Alto, CA) and a voltmeter (34401A, Agilent), which were connected to a personal computer through a GPIB interface. A CCD camera (CV-S3300, JAI Corp., Japan) connected to a framegrabber board (IMAQ PCI-1411, National Instruments Corp., Austin, TX) and controlled by data acquisition software (IMAQ Vision Builder, National Instruments) were used to record the contact area and apex height of the droplets deposited on the QCM. The optical resolution of the setup was 1.4 µm. Diffusive drying experiments with both polished (average surface roughness ∼50 Å) and unpolished AT-cut gold-coated crystals (average surface roughness ∼1.8 µm) were performed at room temperature (22.4-24.3 °C) and 0% RH. The diameter of the crystal was 1 in. and the electrode diameter was 0.5 in. All of the stock solutions used in the experiments were prepared gravimetrically on a microbalance using distilled, filtered water. The water contents of the prepared solutions were also confirmed gravimetrically after baking at 85 °C for more than 48 h. High-purity trehalose dihydrate was purchased from Pfanstiehl (Ferro Pfanstiehl Laboratories Inc., Waukegan, IL). Dextran at different molecular weights, 3-O-methyl-D-glucopyranose (3-OMG), and glycerol were purchased from Sigma (Sigma-Aldrich Corp., St. Louis, MO). Prior to preparation of the solutions, all of the powder chemicals were kept in an 85 °C atmospheric oven for 24 h to remove the residual water. Between experiments, the crystal was removed from its holder, washed with excess amounts of distilled water, and dried by purging with ultradry nitrogen gas. Experiments with different solutions were repeated five to nine times.
Results and Discussions Small volumes (Vi ) 0.2 and 0.4 µL, where Vi is the initial volume) of trehalose, 3-OMG, and dextran solutions at different initial concentrations, Ci, were deposited on a QCM and diffusively dried in a constant humidity environment. 3-OMG was chosen as one of the carbohydrates to be experimented with since it has extensively been used in our preservation research due to its active transport potential through the cell membrane (data not shown). All of the samples, immediately after they were deposited on the QCM crystal, assumed a three-dimensional, hemispherical shape. During drying, depending on the nature of the sample solution (and the concentration of the solutes), the shape of the droplet changed. Here, we use the term “droplet” to describe a three-dimensional
shape, whose thickness changes along the contact surface. As examined in detail below, some solutions, even after drying for extensive periods of time, did not form films and remained as droplets. During the experiments, in addition to frequency and resistance data from the QCM, the droplet apex height and the contact area were also recorded using a CCD camera. Ultrapure water, aqueous salt, and aqueous glycerol solutions were used to analyze and calibrate the response of the QCM. Water Droplets. The frequency, F, and the resistance, R, values recorded by the QCM during the evaporation of a 200 nL water droplet are presented in Figure 1. The water droplet deposited on the crystal displaced air proportional to the droplet contact surface area, Ao, causing an instantaneous drop in frequency, ∆F1, accompanied by an increase in the resistance, ∆R1, given by
∆F1,∆R1 ∝ Ao((Fwηw)1/2 - (Faηa)1/2)
(3)
where the subscripts w and a denote the properties of water and air at the experiment temperature and pressure, respectively. Increasing the sample volume (in the range 0.2 e Vi e 1000 µL) increased ∆R1 and ∆F1 linearly (R 2 ) 0.997) with the contact area (with the square of the contact radius, r) of the deposited droplet (Inserts in Figure 1) since the density-viscosity product for air was negligible when compared to that of water in eq 3 and since the droplets were sufficiently small (it is known that the sensitivity of the QCM crystal decreases with distance from the center32 and for large droplets, the linearity may not hold). The ratio, ∆R1/∆F1 was equal to -3.575, giving an Lu value of 0.039 H, which was well within the manufacturer specified range (0.03 < Lu < 0.04). After the droplet was deposited, both F and R remained constant for a period of time (approximately 75 s for a 200 nL droplet on a polished, cold-coated crystal) even though water continued to evaporate (up to point 1 in Figure 1). This was because the viscosity and the density of water and the contact area remained constant (confirmed by (32) Rodahl, M.; Kasemo, B. Sens. Actuators B 1996, 37, 111.
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Figure 2. Normalized apex height (h/ho) as a function of normalized drying time (t/td) for (A) dextran and (B) trehalose droplets (Vi ) 0.2 µL, T ) 24.5 °C, φ ) 0% RH) deposited on polished QCM crystals. (The images are taken after steady-state conditions are reached at t/td ∼ 5).
video imaging). Given that the penetration depth of the shear wave, δ, into water was less than 300 nm, the decrease in the apex height during this drying regime could not be detected by the QCM and therefore F and R remained constant. The penetration depth of the shear wave is given by30
δ)
( ) ηl πfoFl
1/2
(4)
Depending on the droplet-surface interactions, a drying water droplet undergoes different drying regimes. On a hydrophilic surface, initially the droplet contact area remains constant while the apex height decreases. With decreasing apex height, the contact angle formed between the droplet surface at the contact periphery and the drying surface decreases. At the point where the horizontal component of the surface tension overcomes the frictional resistance between the water droplet and the hydrophilic
surface, the contact area starts to decrease (corresponding to point 1 in Figure 1). Due to the steady decrease in the contact area, both F and R decreased until the droplet evaporated completely. When water droplets were dried on an unpolished crystal, the location of point 1 was further shifted to the right due to the increased frictional force imposed on the liquid by the rougher surface (average surface roughness for the unpolished crystal is ∼1.8 µm, compared to ∼50 Å of the polished crystal). Delayed contact surface area decrease caused the water droplets deposited on unpolished crystals to evaporate significantly faster (∼30%) than those deposited on polished crystals. On the polished crystal, the contact line pinning was destroyed earlier and the droplet kept its hemispherical-like shape throughout the drying process. On the unpolished crystal, however, the contact line stayed stationary longer, the droplet surface area-to-volume ratio increased resulting in faster evaporation (i.e., even though the evaporation flux was
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Figure 3. QCM response to a drying trehalose film (Ci ) 0.46875%, Vi ) 0.2 µL, T ) 24.5 °C, φ ) 0% RH).
the same in both cases, the surface areas during evaporation were different). Since the mass contribution to the surface of the crystal was practically zero throughout the drying process, d(∆F/∆F1)/d(∆R/∆R1) remained constant, and equal to 1. Aqueous Glycerol Droplets. Aqueous glycerol solutions (in the range 0-100% w/w water) were used to quantify the QCM response since their physical properties are well-documented in the literature.33 For glycerol solutions, ∆F1 and ∆R1 were linearly proportional to the contact surface area of the droplet and therefore to the deposited volume (similar to the case with water). When the water content of the glycerol solutions was increased (while keeping the droplet volume constant), ∆F and ∆R decreased linearly (R 2 ) 0.988) with (Fgηg)1/2 (the subscript, g, denotes the properties of glycerol, data is not shown). Optical analysis of the drying droplets confirmed that there was no significant difference among the contact areas of different glycerol solutions of the same volume in the range examined (58.25-100% w/w water). Since the drying glycerol solutions did not contribute to the mass but to the liquid loading of the crystal only, d(∆F/∆F1)/d(∆R/ ∆R1) remained constant during drying on a polished crystal. Shape Change in Droplets of Carbohydrate Solutions During Drying. When a small enough droplet of carbohydrate solution is deposited on a gold-coated crystal surface (such as the quartz crystal of a QCM), it initially assumes a hemispherical shape. Desiccation kinetics of the droplet and its final shape, however, are governed by the environmental conditions, the initial concentration of the carbohydrate in the solution, and the probability of a glassy skin formation on its surface. To establish the conditions that would yield to the formation of a thin film (of uniform thickness over its contact area) by isothermal desiccation for a droplet deposited on a QCM crystal, trehalose and dextran solutions at different initial concentrations were dried in a controlled environment and the droplet shape changes during the course of drying were examined. (33) CRC Handbook of Chemistry and Physics; CRC Press: Boca Raton, FL, 2003.
In Figure 2, changes in the apex heights of dextran (MW ) 37 500 Da) and trehalose droplets of Vi ) 0.2 µL are presented as a function of normalized drying time, td
[h1 ∂h∂t ]
td ) -
-1
t)0
(5)
Note that the apex height is normalized with respect to the initial apex height, ht)0. In each experimental group, there are five to nine experiments and the standard deviations are less than the sizes of the symbols. At 30% dextran concentration (Figure 2A), h/ht)0 initially decreased in a linear fashion and then slowed and reached a plateau value, while the contact area of the droplet remained unchanged throughout the drying process (data not shown). The onset of deviation from the linear apex change regime (t/td ∼ 0.25) corresponded to the formation of a glassy skin on the surface of the droplet (confirmed by physically touching the surface of the droplet by a needle). The final shape of the 30% dextran droplet (at t/td ∼ 5) is shown in Figure 2A-IV. Note that the droplets were dried on gold-coated QCM quartz crystal surfaces and that the images were taken at an angle; thus the images also included the reflections of the droplets. Similarly, for the 15% dextran solution, deviation from the linear apex height change regime was observed at (t/td ∼ 0.25); however, it was not as strong as that in the 30% dextran solution. Interestingly, the apex height later increased, reached a local maximum, and gradually decreased toward its final plateau value. This response might be caused by the mechanical collapse of the skin close to the periphery of the droplet (due to the continuous decrease of the volume under the skin by the evaporation of water), pushing the center upward.23 This behavior also confirmed that water continued to evaporate despite the formation of the skin (the skin surface area was larger than necessary to cover the remaining volume of the solution underneath). A similar behavior was observed for the 7.5% dextran solution, this time with a local maxima followed by a lower plateau and a transition toward a higher plateau value. As shown by these images (Figure 2A-II-IV), high initial concentration dextran
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Figure 4. QCM response of trehalose, dextran, and 3-OMG films during drying (A) Vi ) 0.2 µL, (B) Vi ) 0.4 µL (Ci ) 0.46875-3.75%, Vi ) 0.2 µL, T ) 24.5 °C, φ ) 0% RH).
droplets did not form thin films by isothermal drying due to the mechanical resistance of and the instabilities invoked by the formation of a glassy skin on the surface. Instead, after going through various drying regimes, they remained as droplets of nonuniform geometry. At lower dextran concentrations however (Ci e 3.75%), thin films were eventually formed (Figure 2A-I). At these low concentrations, throughout the drying process the apex height decreased linearly and most of the water inside the droplet evaporated before a film was formed on the surface of the QCM crystal. For the trehalose droplets (Figure 2B), h/ht)0 did not have local maxima or minima and decreased continuously, at variable rates (irrespective of Ci). As a function of the initial concentration, Ci, the contact area changed as well
(data not presented). For a 30% trehalose droplet, there was a single drying regime with gradual decrease in apex height and contact area. With decreasing initial concentration of trehalose in the solution, three different evaporation regimes appeared. In each regime the time rate of change of either h/ht)0 or the contact surface area, alternatively, was higher than its value in the previous regime (i.e., the rate of apex height decrease was higher when the rate of contact diameter decrease was lower). The absence of apex height increase in trehalose (as opposed to that in dextran) was attributed to the differences in the mechanical properties of the skins formed by these carbohydrates (i.e., the glassy skin formed by trehalose was stiffer than that of dextran and therefore resisted buckling). This observation is in accord with the
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previously published results from our group, where very different cracking patterns were observed on the surfaces of the trehalose and dextran droplets dried for very long periods of time (td > 1000 h).21 QCM Analysis of Films of Carbohydrate Solutions. High initial concentration solutions (Ci > 3.75%) assumed three-dimensional (3-D) shapes during drying (Figure 2A-II-IV) and did not form films and therefore were not suitable for QCM analysis. The final dried shapes of the droplets depended on Ci as well as on the mechanical properties of the skin formed by that particular carbohydrate. With lower initial concentration solutions (Ci e 3.75%), the apex height of the droplet decreased continuously and a film was formed before the surface of the droplet reached the critical solute concentration required for the formation of a glassy skin. A film has uniform thickness throughout and therefore eliminates artifacts in QCM analysis that may result from the nonuniform thickness over the contact surface. Penetration depths of the QCM shear waves were approximately 270 nm for a 0.5% w/w dextran solution and increased exponentially with increasing concentration. It was therefore reasonable to assume that during the desiccation experiments, with increasing viscosity of the solution, the penetration depth of the shear waves could easily reach the surface of the drying product. If the shape of the droplet was irregular (as would be the case with high Ci), then the distribution of mass and liquid loading per unit area of the crystal would be spatially nonuniform causing artifacts in the measurement. When a low concentration carbohydrate solution was deposited on the surface of the QCM, F and R changed rapidly (Figure 3) in the amounts predicted by eq 3. Initially (in regime 1), due to the high h/δ ratio, and the fact that a glassy skin did not form, no significant change in F or R could be observed (actually, even though R remained constant, F decreased by approximately 1-1.5% of ∆F1). During this regime Ao remained constant and the time rate of change of apex height decrease was at a maximum. The onset of contact area shrinkage marked the second drying regime, where R and F started to decrease. Note that the slopes of the F and R curves were determined by the relative contributions from the decrease in the contact area and the increase in the viscositydensity product. At very low concentrations (Ci ∼ 0.46875% w/w), ∆F and ∆R decreased significantly up to a point marking the onset of the third regime, where the effect of the increase in the viscosity-density product in the shear boundary layer was more significant than that of the decrease in the contact area. In the third regime (regime 3 in Figure 3), ∆F and ∆R increased exponentially up to the liquid loading limit of the QCM. At this limit value, QCM response started to change from liquid loading to mass loading;34 ∆R started to decrease exponentially toward zero, while ∆F started to decrease (with ∆Fl f 0), and converged toward its final ∆Fm value. In Figure 4, variation of ∆F/∆F1 with ∆R/∆R1 for Vi ) 0.2 µL and Vi ) 0.4 µL droplets are presented for different Ci trehalose, 3-OMG, and dextran solutions. As shown, during drying initially, there was only liquid loading (and therefore ∆F/∆R was constant) and all of the curves ran parallel to that for glycerol solutions. When the liquid loading limit was reached, ∆R/∆R1 started to decrease while ∆F/∆F1 values slowly converged toward their final equilibrium values governed by eq 1 (indicating pure mass loading). Similar behaviors were observed for both the Vi (34) Kanazawa, K. K. Faraday Discuss. 1997, 107, 77.
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Figure 5. ∆Rmax/∆R1 for Ci ) 0.93725% carbohydrate solutions.
) 0.2 µL and the 0.4 µL experimental groups showing the insensitivity of the results to the initial volume of the sample. The transition in the QCM response from liquid to mass loading during drying of a carbohydrate solution presented a unique opportunity to compare the mechanical properties of the films of different carbohydrate solutions. For all of the concentrations experimented with, the maximum value of ∆R/∆R1 was the highest for 3-OMG followed by dextran (both for 3000 and 37500 Da) and the trehalose films (Figure 5). A higher ∆R/∆R1 value corresponding to the same ∆F/∆F1 indicated that the contribution to the frequency shift was mostly from the liquid loading (mainly, the increase in the viscosity-density product) rather than the mass loading. The higher the mass loading contribution to the frequency shift, on the other hand, the stiffer is the film, since the stiffer it gets, the more it starts to behave like an elastic solid rather than a viscous liquid. These results indicate that the stiffness of the trehalose films formed by isothermal desiccation is higher than those of dextran and 3-OMG supporting the observations from the optical measurements in this research; lower stiffness of the dextran skin caused instabilities during drying (and buckled), yielding to differences in the final shapes of the droplets. Our previous research also supports this finding that the crack patterns on trehalose and dextran droplets are different. Recent neutron scattering experiments also support these findings; when compared to other disaccharides (namely, sucrose and maltose), the glass of trehalose is more rigid.35 The order of the ∆Rmax/∆R1 values (Figure 5) is not the same as the order of the glass transition temperatures for the sugars experimented with (Tg,dextran > Tg,trehalose > Tg,3-OMG). However, it is well-known that the bioprotective capacity of trehalose (against freeze and desiccation damage) is higher than that of any other carbohydrate tested to date. This may present additional evidence that a higher glass transition temperature is a necessary but not the sufficient condition for biopreservation efficiency. Conclusions Desiccation and vitrification kinetics of a carbohydrate solution depends on its physicochemical properties,23 the initial concentration of the carbohydrate in the solution and the interaction of the solution with the surface it is drying on.36 At high initial concentrations, overall desiccation rate of the solution is limited by the diffusion of (35) Magazu, S.; Maissano, G.; Migliardo, F.; Mondelli, C. J. Phys. Chem. B 2004, 108, 13580. (36) Deegan, R. D.; Bakajin, O.; Dupont, T. F.; Huber, G.; Nagel, S. R.; Witten, T. A. Phys. Rev. E 2000, 62, 756.
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water in the solution (as opposed to its diffusion in the surrounding environment). At the early stages of drying, a skin is formed on the surface of the droplet introducing additional resistance to the droplet shape change,36 indirectly affecting the drying rate, the homogeneity within, and the final shape of the droplet. Even though the skin does not offer much resistance to evaporation, it resists the contraction of the droplet in response to a decrease in its volume. With continuing evaporation and decrease of droplet volume the skin becomes increasingly larger than needed to cover the volume underneath and compressive mechanical stresses are formed. These compressive stresses induced in the skin layer results in either buckling of the skin if it is compliant (for example in dextran solutions) or ultimately cracking if it is brittle (for example in extensively dried trehalose solutions). Skin formation is directly related to the initial concentration of the carbohydrate in the solution and plays a major role in determining the final shape of the dried droplet (Figure
Aksan et al.
2). The surface-droplet interactions also play important roles such that with increased friction, contact line movement is delayed increasing the surface area-tovolume ratio, therefore increasing the evaporation rate. Using low initial concentration solutions and therefore ensuring uniform film formation (rather than irregularly shaped droplets) during isothermal desiccation, it was possible to explore the vitrification characteristics of different carbohydrate solutions. It was demonstrated that using QCM analysis, it was also possible to compare the mechanical properties of very thin films of carbohydrate solutions. Acknowledgment. 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. LA047760Y
