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Protein Glasses
Journal of Pharmaceutical Sciences xxx (2016) 1-9
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Pharmaceutical Biotechnology
Hydrogen Bonding Interactions and Enthalpy Relaxation in Sugar/Protein Glasses €tte Oldenhof 2, Harald Sieme 2, Willem F. Wolkers 1, * Bulat Sydykov 1, Harrie 1 2
€t Hannover, Hannover, Germany Institute of Multiphase Processes, Leibniz Universita Clinic for HorseseUnit for Reproductive Medicine, University of Veterinary Medicine Hannover, Hannover, Germany
a r t i c l e i n f o
a b s t r a c t
Article history: Received 7 October 2016 Revised 31 October 2016 Accepted 1 November 2016
In this study, hydrogen bonding interactions and enthalpy relaxation phenomena of sugar and sugar/ protein glasses have been studied using Fourier transform infrared spectroscopy and differential scanning calorimetry. The sugar OH band in Fourier transform infrared spectra was used to derive the glass transition temperature, Tg, and the wavenumber-temperature coefficient (WTC) of the OH band. A study on mixtures of sucrose and albumin revealed that the glass transition temperature and strength of hydrogen bonds increased with increasing percentages of albumin. WTCg and Tg derived from sucrose/ albumin glasses showed a negative linear correlation. The Lu-Weiss equation was used to fit Tg data of sucrose/albumin mixtures. An inflection point was observed at a 1:1 mass ratio, which coincided with an inflection of the OH-stretching band denoting a change in hydrogen bonding interactions. Enthalpy relaxation, which is seen as an endothermic event superimposed on the glass transition in differential scanning calorimetry thermograms, increases with increasing storage temperature. Activation energies of enthalpy relaxation of sucrose and sucrose/albumin glasses were determined to be 332 and 236 kJ mol1, respectively. Addition of albumin to sucrose increases the Tg, average strength of hydrogen bonding, heterogeneity, and the enthalpy relaxation time, making the glass more stable during storage at room temperature. © 2016 American Pharmacists Association®. Published by Elsevier Inc. All rights reserved.
Keywords: amorphous calorimetry (DSC) FTIR glass proteins stability
Introduction Sugars, such as sucrose and trehalose, can protect biological materials during cryopreservation and lyophilization. Protective properties of sugars have been attributed to their ability to form hydrogen bonds and facilitate glass formation during dehydration.1,2 A glass is a highly viscous amorphous state in which molecular mobility is decreased, and damaging reactions are slowed down.2-4 Moreover, it functions as a matrix in which cellular and biomolecular structures are embedded and prevented from interacting with one another. Typical formulations used for preservation of pharmaceutical or biological materials are composed of sugars and biopolymers. Glasses composed of sugars and proteins have been implicated for dry preservation of cells.5,6 In addition to the composition, the storage temperature and relative humidity conditions are the main factors that affect molecular mobility and stability
* Correspondence to: Willem F. Wolkers (Telephone: þ49 511 762 19353; Fax: þ49 511 762 19389). E-mail address: [email protected] (W.F. Wolkers).
in the glassy state. Uptake of water has a plasticizing effect on the glass transition, which means it decreases Tg and increases molecular mobility, leading to faster degradation and crystallization processes.7 The dependence of Tg on the water content can be determined experimentally or predicted using the Gordon-Taylor equation which describes the Tg of miscible binary mixtures.7,8 Amorphous materials are in a nonequilibrium state at temperatures below Tg and have a higher specific volume and enthalpy compared to the equilibrium state. The inherent tendency of glassy materials to transition into the equilibrium state is referred to as physical aging or structural relaxation.9,10 The rate and extent of physical aging affect the longevity of preserved materials in glasses. Especially, occurrence of crystallization may cause damage to materials preserved in a glassy state. The relaxation rate in the glassy state depends on the degree of molecular mobility and the difference between the storage temperature and Tg. Differential scanning calorimetry (DSC) is one of the most widely used methods to measure glass transitions of amorphous compounds. The glass transition is evident as a change in heat flow during heating or cooling. Structural relaxation of glasses can
http://dx.doi.org/10.1016/j.xphs.2016.11.003 0022-3549/© 2016 American Pharmacists Association®. Published by Elsevier Inc. All rights reserved.
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be observed as an endothermic peak superimposed on the glass transition.11 If enthalpy relaxation is plotted versus storage duration, the rate of relaxation can be fitted using the KohlrauschWilliams-Watt equation.12 This in turn gives insights into molecular mobility in glasses,11 and stability of embedded materials.13 Fourier transform infrared (FTIR) spectroscopy also has been used to study glass transitions, particularly of carbohydrates. Glass transitions of sugars can be determined by monitoring the position of the OH-stretching vibration band (nOH) arising from sugar OH groups versus the sample temperature.14,15 When nOH is plotted versus the sample temperature, the glass transition is visible as a change in slope of nOH versus temperature above and below the glass transition. The slope is indicative of the strength of the hydrogen bonding network in the glassy or liquid state. The aim of the present study was to assess physical characteristics of sugar and sugar/protein glasses with special emphasis on factors affecting Tg, hydrogen bonding interactions, and enthalpy relaxation. It was hypothesized that adding albumin to sugar glasses increases Tg and decreases molecular mobility. Both FTIR spectroscopy and DSC were used to study Tg, molecular interactions, and enthalpy relaxation characteristics. Sugar/albumin glasses were studied at different mass ratios. The effect of water content on Tg and enthalpy relaxation at different storage temperatures was studied. Data on enthalpy relaxation during storage were fitted to derive parameters describing temperature-dependent molecular mobility in glasses. Materials and Methods Preparation of Carbohydrate Glasses Sugar glasses were prepared from glucose, sucrose (both from Carl Roth, Karlsruhe, Germany), and trehalose (Cargill, Minneapolis, MN). Sugar solutions of 20-50 mg$mL1 were prepared using distilled water. In addition, mixtures of albumin (bovine serum albumin fraction V, pH 7.0; Serva, Heidelberg, Germany) and sucrose at different sucrose/albumin mass ratios were prepared. For FTIR analyses, sugar glasses were prepared by drying 10-20 mL sugar or sugar/albumin solution on infrared transparent CaF2 windows (25 2 mm; Korth Kristalle GmbH, Altenholz, Germany). This was performed in a box purged with dry air (relative humidity less than 3%). Before measurements, residual water was removed by heating the sample (100 C for at least 3 min). The absence of water after heating was verified from the disappearance of the HOH scissoring band at ~1645 cm1. Sugar glasses for DSC analyses were prepared by freeze-drying. A volume of 1 mL of sugar or sugar/albumin solution was added in freeze-drying vials (2R injection vials; Landgraf Laborsysteme, Langenhagen, Germany), and vials were placed on the temperaturecontrolled shelves of a lyophilizer (Virtis Advantage Plus Benchtop freeze dryer; SP Scientific, Warminster, PA). Samples were cooled from 20 C down to 30 C at 1 C min1 and kept at 30 C for 1 h. Primary drying was performed at a temperature of 30 C and a pressure of 60 mTorr, for 15 h. Then, the shelf temperature was increased to 40 C, at 0.1 C min1 while maintaining a pressure of 60 mTorr. Samples were maintained at 40 C for 1 h, after which secondary drying was performed at a temperature of 20 C and pressure of 10 mTorr for 6 h. After freeze-drying, freeze-dried material was directly used for DSC analysis or processed for storage. Samples with different water contents were obtained by placing pans with freeze-dried material in containers with different relative humidity before sealing them. A relative humidity of 35% and 75% was obtained using saturated salt solutions of CaCl2 and NaCl, respectively.
Fourier Transform Infrared Spectroscopy Studies Infrared spectra were recorded using a Perkin-Elmer 100 FTIR spectrometer (Perkin Elmer, Norwalk, CT), equipped with a triglycine sulfate detector, a temperature-controlled sample holder connected to a heating device (Harrick Scientific Products, Pleasantville, NY), and a Linkam pump system for using liquid nitrogen as a coolant (Linkam Scientific Instruments, Tadworth, Surrey, UK). The optical bench was continuously purged with dry air from an FTIR purge gas generator (Whatman, Clifton, NJ). Spectra acquisition parameters were as follows: 4 cm1 resolution, 4 co-added interferograms, and 4000-900 cm1 wavenumber range. Sugar glasses prepared on a CaF2 window, as described above, were covered with a second window while having a Teflon spacer between them to avoid sticking. This was mounted in the sample holder, residual water was removed as described above, and the sample temperature was decreased to 30 C, at 1 C min1. Then, the temperature was increased to temperatures up to 180 C, at 1 C min1, while acquiring spectra every 30-60 s. A T-type thermocouple (Fluke, Everett, WA) was used to monitor the sample temperature. In addition to using Perkin-Elmer software, spectral analysis and display were carried out using Omnic (ThermoElectron Corporation, Waltham, MA) as well as Wolfram Mathematica software (Wolfram Research, Champaign, IL). Melting of sugar glasses was monitored by following the position of nOH around 3300 cm1 versus the sample temperature, as previously described in detail.14,15 In short, the 3600-3000 cm1 spectral region was selected and normalized, after which the band position was calculated as the average of the spectral positions at 80% of the full peak height. Plots were constructed in which nOH was plotted as a function of the sample temperature, and linear regression lines in both the liquid and glassy state regions of the plot were added. The glass transition temperature (Tg) was determined as the intersection point of these 2 regression lines, whereas wavenumber-temperature coefficients (WTCg and WTCl) refer to the slopes of the regression lines. Differential Scanning Calorimetry DSC measurements were taken using a Netzsch DSC 204F1 Phoenix instrument (Netzsch-Geraetebau GmbH, Selb, Germany). Calibration was performed using adamantane, bismuth, indium, zinc, selenium, and cesium chloride, according to the instructions provided by the manufacturer. Approximately 10 mg of freezedried sample, prepared as described above, was added into a 25-mL aluminum pan. After sealing pans hermetically, the fresh sample weight was determined using a microbalance. Sealed pans with samples were either directly used for DSC analysis or stored for different durations (up to ~1 week) at different temperatures (1 C50 C) for analyses of glass characteristics during aging. With DSC measurements, an empty pan was used as a reference sample. In order to study glass transitions, samples were cooled to 50 C with 10 C min1 and held there for 5 min, after which they were heated to 150 C at 10 C min1, while monitoring the heat flow. Thermal events were determined from the obtained thermograms, using Netzsch software. Dry weights of samples were determined after DSC analyses, after perforating the pans and overnight incubation in an oven set at 80 C. Water contents were determined by comparing the fresh and dry weight:
WC ¼
½ðFW þ PÞ P ½ðDW þ PÞ P ½ðDW þ PÞ P
(1)
FW and DW represent the fresh and dry weights of the sample, respectively, P is the weight of the pan (all in grams), and WC is the water content (in g H2O per g DW).
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The glass transition temperature (Tg) was determined as the midpoint of the temperature range, over which the change in specific heat coinciding with the transition from glassy to liquid state occurred, whereas DCp is the change in heat flow at Tg divided by the heating rate. Enthalpy relaxation (or enthalpy recovery) was observed as an endothermic event occurring superimposed on top of the glass transition and quantified as the area under the endothermic peak associated with Tg, according to Liu et al.16 as described below. Glass Transition Models of Binary Mixtures Different models were tested for predicting the dependence of the glass transition temperature on the composition of binary mixtures, including the Gordon-Taylor and Lu-Weiss models. The Gordon-Taylor model was originally derived for polymer blends and copolymers assuming that the volumes of the 2 components are additive and there are no specific interactions between the components17:
x1 Tg;1 þ kð1 x1 ÞTg;2 Tg ¼ x1 þ kð1 x1 Þ
(2)
Here x1 and Tg,1 are the mass fraction and the glass transition temperature (in Kelvin) of the first component, whereas Tg,2 is of the second component. The parameter k is defined as r1Da2/r2Da1, where Da is the change in coefficient of thermal expansion at Tg, and r1 and r2 represent the density of the first and second component. Actually, k can be used as a parameter to describe interactions between components in a binary mixture,18 such as hydrogen bond interactions in water-carbohydrate mixtures.19 The Lu-Weiss model describes the glass transition temperature behavior for miscible binary polymer blends taking into account interactions (e.g., ionic interactions, hydrogen bonding) between components20:
Tg ¼
x1 Tg;1 þ k$ð1 x1 ÞTg;2 þ Ax1 ð1 x1 Þð1 þ dð1 x1 ÞÞ x1 þ k$ð1 x1 Þ
(3)
In addition to the parameters described for the Gordon-Taylor equation, k, A, and d are used as fitting parameters. k represents the ratio of the change of heat capacities at the Tg of the second versus the first component, A is a parameter describing the interactions, and d contains information about the linear dependency of k and densities and molecular weight ratios of the 2 components.
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Here DCp (in J$g1$K1) is the change in heat capacity at Tg, and T (in Kelvin) represents the storage temperature. Enthalpy relaxation during storage then can be fitted using:
( DHrelaxation ðtÞ ¼ DH∞
"
b #) t 1 exp t
(5)
This requires 2 fitting parameters, namely t which is the mean relaxation time (in seconds) and b (which is 0 and 1) representing a nonexponential parameter describing the distribution of relaxation times. b is equal to 1 in case of a single relaxation time whereas smaller values for b are indicative of a wider distribution of t. Arrhenius plots of relaxation time versus storage temperature were fitted using:
ln t ¼
Ea 1000 þ lnt0 T R
(6)
where R is the gas constant (8.314 J mol1 K1), the mean relaxation time t is given in seconds, and storage temperature T in Kelvin. The activation energy Ea (in kJ mol1) follows from the slope of the linear plot, whereas t0 can be calculated from the intersection point. Results Infrared Spectra and Temperature Dependence of nOH In Figure 1, typical infrared spectra of sugar glasses, prepared from glucose, sucrose, and trehalose, are shown. In addition, spectra of crystalline sucrose and albumin are shown. Residual water is evident as a band around 1650 cm1 arising from HOHscissoring vibrations. OH-stretching vibrations resulting from sugars can be found in the 3600-3000 cm1 region. It can be seen that absorbance bands in amorphous sucrose are broader compared to crystalline sucrose. This is explained by the more variable lengths and orientations of hydrogen bonds as present in a glass. Also, the peak position of the OH band of crystalline sucrose is shifted to a lower wavenumber, indicating stronger hydrogen bonds compared to those in the amorphous state. The 3000-2800 cm1 region contains CH-stretching vibrations arising from the
Enthalpy Relaxation DSC pans with sucrose and sucrose/albumin (1:1 mass ratio) glasses were stored for different durations at different temperatures as described above. Enthalpy relaxation values were derived from DSC thermograms and plotted versus the storage time. Enthalpy relaxation of stored sucrose/albumin glasses was determined by subtracting the thermogram of a nonaged sample. In case of sucrose glasses, the thermogram was extrapolated from above Tg to lower temperatures without the endothermic glass relaxation peak, and this curve was subtracted from the original thermogram exhibiting enthalpy relaxation.11 To derive parameters describing molecular mobility in a glass, the dependence of the enthalpy relaxation (DHrelaxation in J g1) on the storage duration (t in seconds) was fitted using the KohlrauschWilliams-Watt equation. First, the maximum possible enthalpy recovery DH∞ at a given storage temperature is described by:
DH∞ ¼ DCp Tg T :
(4)
Figure 1. Infrared absorption spectra of sugar glasses, albumin (BSA), and crystalline sucrose. nOH is visible around 3300 cm1, and the CH2-stretching region is from 3000-2800 cm1. The amide-I and amide-II bands of BSA are visible at 1650 and 1550 cm1, respectively.
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carbohydrate methyl groups. The 1500-1000 cm1 region is referred to as the “fingerprint” region which, in case of sugars, contains absorbance bands arising from CH-deformation, C¼O stretching, and OH-bending vibrations. The spectrum of albumin displays 2 main bands at around 1650 and 1550 cm1, which can be attributed to the amide I and amide II band, respectively.21 Albumin also has an OH band arising from intrinsic OH groups. However, the protein OH band is relatively weak compared to that of the sugars, because sugars have a relatively high number of OH groups.22 The temperature dependence of nOH position (~3300 cm1) was studied to determine glassy behavior and glass transition temperatures (Fig. 2a). With increasing sample temperature, molecules will have more thermal energy, which leads to a reduced level of hydrogen bonding and a shift of the nOH to a higher wavenumber. At the glass transition temperature, there is a change of slope in the nOH wavenumber position versus temperature plot. The Tg was derived from the intersections of linear regression lines in the glassy and liquid regions, below and above Tg, and was determined to be 108 ± 3 C for trehalose, 58 ± 1 C for sucrose, and 30 ± 2 C for glucose (Fig. 2b). The slopes of the regression lines (WTC) are a measure of the strength of the hydrogen bonding network; the higher the value of WTC, the weaker the hydrogen bonds. In the glassy state, the WTC value is lower (0.205-0.290 C cm1) as compared to the liquid state (0.518-0.548 C cm1), indicating stronger hydrogen bonds. In the liquid state, no large differences were found between WTC values of the studied materials. In the glassy state, however, the WTC for glucose was lower as compared to values determined for sucrose and trehalose. This most likely is due to the smaller size of glucose compared to that of disaccharides.
hydrogen bonds (i.e., decreased WTCg). For sucrose/albumin mixtures, with relative sucrose content up to ~0.5 g/g, a negative linear correlation was found when WTCg was plotted versus Tg (Fig. 4a). This is different from the positive linear correlation that was found when plotting data obtained for glasses made from pure sugars with different molecular weights.15 It should be noted that albumin also has an OH band arising from intrinsic OH groups (Fig. 1). However, in sucrose/albumin mixtures, the OH band is dominated by the OH band of sugars. At higher albumin contents, however, the band may also comprise a contribution of the albumin OH band. The band position of the protein OH groups hardly changes with temperature and therefore the glass transition of albumin alone cannot be measured by this FTIR method, because in contrast to sugar OH groups, protein OH groups do not directly interact with the glassy matrix. The glass transition can only be detected if sugars are present and can be determined at sucrose contents as low as 0.1 g/g. When Tg was plotted versus the relative sucrose content in the sucrose/albumin mixture, an inflection point is seen at relative sucrose contents of 0.4-0.6 g/g, below which a linear correlation can be seen (Fig. 4b). The Tg of pure albumin was estimated to be 178 C via extrapolation of data in the linear region (below 0.4) toward 0. The Gordon-Taylor equation, which assumes absence of specific interactions in a mixture, could not be used to fit the relation between Tg and the relative sucrose content in sucrose/ albumin mixtures. This is likely due to hydrogen bonding interactions between the components, as evident from plots of nOH versus the relative sucrose content (Fig. 4b). Data were successfully fitted with the Lu-Weiss equation, which takes into account interactions between components in a mixture (with k ¼ 0.162, A ¼ 149, d ¼ 3.96).
Glass Transition Temperatures and WTC of Sucrose/Albumin Glasses Glasses prepared using different sucrose/albumin mass ratios were subjected to FTIR analysis, and nOH was monitored versus the sample temperature in order to derive Tg and WTC values (Fig. 3a). In Figure 3b, it can be seen that an increased relative percentage of albumin (i.e., lower sucrose/albumin mass ratio value) resulted in an increase in the glass transition temperature and strength of
Effect of Water Content on Tg and DCp of Sucrose and Sucrose/ Albumin Glasses DSC was used to analyze effects of the sample water content on characteristics of both sucrose and sucrose/albumin (1:1 mass ratio) glasses. Tg was determined as the onset temperature at which the change in specific heat coinciding with the transition
Figure 2. FTIR was used for studying temperature-dependent glass-to-liquid transitions in sugar glasses. This was performed for the disaccharides trehalose (white circles, bars without filling) and sucrose (grey circles, bars with diagonals), as well as monosaccharide glucose (black circles, bars with diamonds). Infrared spectra were collected during heating, and the position of nOH was determined and plotted versus the sample temperature (a). The glass transition temperature (Tg) was determined as the intersection point of the regression lines in the liquid state and in the glassy state (b), whereas WTC represent the slopes of the regression lines in both states (c; WTCg and WTCl). Mean values ± standard deviations are presented, determined from 6 independent experiments.
B. Sydykov et al. / Journal of Pharmaceutical Sciences xxx (2016) 1-9
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Figure 3. FTIR analyses of glasses prepared using different mass ratios of sucrose (SUC) and albumin (BSA). In panel a, plots are shown on nOH versus the sample temperature; for SUC/BSA glasses prepared at mass ratios (w/w) of 9:1 (white circles), 6:4 (grey circles), and 2:8 (black circles). In panel b, plots are shown on the Tg (white squares) as well as the WTCg (black squares) versus the sucrose/albumin mass ratio.
from glassy to liquid state occurred, whereas DCp was defined as the change in heat flow at Tg divided by the heating rate (Fig. 5a). It was found that Tg decreases with increasing water content, both for sucrose and sucrose/albumin glasses, which can be attributed to the plasticizing effect of water. At water contents lower as 0.05 g water per gram dry weight, the Tg of sucrose is higher as compared to sucrose/albumin glasses. At higher water contents, a glass transition of 27 C (>0.05 g H2O g DW1) was determined for sucrose glasses, which did not further decrease. This refers to Tg’, which is the Tg of the maximally freeze-concentrated amorphous phase. For sucrose/albumin glasses, Tg values continued to decrease. The DCp values associated with the glass transition were found to be greater for sucrose glasses compared to sucrose/ albumin mixtures at water contents below 0.05 g H2O g DW1. For sucrose glasses, however, DCp decreased with below 0.05 g H2O g DW1, whereas DCp of sucrose/albumin glasses remained constant as a function of the water content. It should be noted that the heat flow was not calibrated for Cp measurements using sapphire as a reference standard. The DCp values were approximated directly from the heat flow, which causes inaccuracies in the reported values.
Enthalpy Relaxation in Sucrose and Sucrose/Albumin Glasses In sugar glasses, there is molecular mobility and enthalpy relaxation, which is seen as an endothermic event superimposed on the glass transition in DSC thermograms (Fig. 5a). For sucrose and sucrose/albumin glasses (1:1mass ratio), DHrelaxation was determined as the area of this peak and monitored during storage at different temperatures. Representative DSC thermograms are shown in Figures 6a and 6b, and DHrelaxation versus storage duration is plotted in Figures 6c and 6d. At a given time point, enthalpy relaxation and thus glass molecular mobility were determined to be higher with increasing storage temperature, with larger effects for sucrose glasses as compared to sucrose/ albumin glasses. Data were fitted using the Kohlrausch-WilliamsWatt equation, to derive the maximum possible enthalpy recovery DH∞, the mean relaxation time t, and parameter b (stretch power) describing the distribution of relaxation times. Data are summarized in Table 1. DH∞ was found to increase with decreasing storage temperature, and DH∞ was found to be lower for sucrose/albumin glasses compared to sucrose glasses. Values for t increased with decreasing storage temperature for both
Figure 4. In panel a, a correlation plot is shown on WTCg values in the glassy state versus the Tg, determined using FTIR, for glasses prepared from sucrose/albumin mixtures (black squares; 1:10-9:10 mass ratios). In addition, data are presented for glasses made from various oligo- and polysaccharides (white squares: glucose, octulose, sucrose, umbelliferose, trehalose, raffinose, glucan, and dextran-T40, adapted from the study by Wolkers et al.15). In panel b, the Tg (black circles) and the nOH peak position at 25 C (white circles) are plotted versus the relative sucrose content in sucrose/albumin glasses. The dependency of Tg on the relative sucrose content was fitted using the Lu-Weiss equation.
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Figure 5. DSC was used to study the glassy behavior of sucrose (white squares) and sucrose/albumin (1:1 mass ratio; black squares) glasses of different water contents. In panel a, representative DSC thermograms (heating scans) are presented to illustrate how Tg, DCp, and DHrelaxation were derived. In panels b and c, Tg and DCp are plotted versus the sample water content. Exothermic events are oriented downward.
Figure 6. DSC was used to study enthalpy relaxation behavior of sucrose (a and c) and sucrose/albumin (1:1 mass ratio; b and d) glasses during storage at different temperatures. In panels a and b, representative thermograms are shown to illustrate differences. Exothermic events are oriented downward. In panels c and d, DHrelaxation is plotted as a function of the storage duration, for sucrose (white squares: 1 C, light grey squares: 22 C, dark grey squares: 30 C, black squares: 38 C) and sucrose/albumin glasses (white squares: 1 C, light grey squares: 22 C, dark grey squares: 37 C, black squares: 52 C), respectively. Data were fitted using the Kohlrausch-Williams-Watt equation, for deriving parameters describing molecular mobility in glasses.
B. Sydykov et al. / Journal of Pharmaceutical Sciences xxx (2016) 1-9 Table 1 Parameters Describing Molecular Mobility in Sucrose and Sucrose/Albumin Glasses (1:1 Mass Ratio) Stored at Different Temperatures, as Derived From Fitting the Enthalpy Relaxation Data Obtained From DSC Measurements Versus Storage Duration, Using the Kohlrausch-Williams-Watt Model Compound
Tstorage ( C)
DH∞ (J kg1)
t (d)
b
Sucrose
38 30 22 1 52 38 22 1
11.34 18.70 26.06 45.37 5.77 10.97 16.92 24.73
1.62 43 99 52614200 0.92 49 495860 5089760
0.43 0.31 0.44 0.23 0.40 0.26 0.15 0.20
Sucrose/albumin
Compound
Tstorage ( C)
Tg ( C)
t (d)
b
Trehalose Sucrose
40 50
113 69.4
5565186 12
0.188 0.199
Parameters include the following: the maximum possible enthalpy recovery
DHrelaxation (in J kg1), the mean relaxation time t (in d), and parameter b describing the distribution of relaxation times (equals 1 in case of a single relaxation, where lower values are indicative of a wider distribution). For comparison, data of freeze-dried sucrose and trehalose from Liu et al.23 are included (last 2 rows).
sucrose and sucrose/albumin glasses. Values for b were lower for sucrose/albumin glasses, indicating a wider distribution of the relaxation time (i.e., less homogenous glass) as compared to sucrose glasses. In order to visualize differences between temperaturedependent glass relaxation parameters in sucrose and sucrose/ albumin glasses, plots were constructed in which the natural logarithm of t was plotted versus the difference between Tg and Tstorage (Fig. 7). This gives a linear correlation, with sucrose glasses exhibiting higher values for ln(t) and a steeper slope compared to sucrose/albumin glasses. An Arrhenius plot (inset in Fig. 7) was used to derive activation energies. Activation energies of enthalpy relaxation for sucrose and sucrose/albumin glasses were determined to be 332 and 236 kJ mol1, respectively.
Figure 7. Parameters describing molecular mobility in glasses were determined by fitting DSC data on enthalpy relaxation versus storage, both for sucrose (white squares) and for sucrose/albumin glasses (1:1 mass ratio; black squares). The natural logarithm of the relaxation time t is plotted versus the temperature stored above Tg. In the inset figure, data are presented in Arrhenius plots for deriving activation energies.
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Discussion FTIR and DSC were used to study physical properties of sucrose and sucrose/albumin glasses. Glasses composed of sugars and proteins have been implicated for dry preservation of cells.5,6 Albumin has been used as protein in these cases. Biological glasses in anhydrobiotic organisms also comprise of a mixture of sugars and proteins such as late embryogenesis abundant proteins that protect during drying. Among other protective functions, these proteins form a glass together with sugars and increase the Tg and molecular packing density of the cytoplasmic glassy matrix in desiccation-tolerant cells.24 The Tg of anhydrous sucrose/albumin glasses is dependent on the mass fraction of sucrose, and a plot of Tg versus sucrose content shows a sigmoidal-shaped curve (Fig. 4b). A similar sigmoidalshaped curve has been observed for maltose/dextrin and glucose/ dextrin glasses25 as well as for mixtures of polyvinyl chloride and phthalates.26 According to Kawai and Hagura,25 binary systems are composed of different regions with different microcompositions. At high relative sucrose content, sucrose predominantly interacts with other sucrose molecules, whereas at sucrose contents below 0.5 g/g, sugars form hydrogen bonds with albumin. Different models can be used to fit Tg data of binary mixtures.27 The Gordon-Taylor equation/model is most commonly used. In cases where there are multiple regions with different microcompositions, such as in binary mixtures, an inflection point is often observed above and below a critical composition which can be described using the models of Braun and Covacs28 and Scandola et al.29 The Kwei equation30 takes into account hydrogen bond interactions between components. The Lu-Weiss equation,20 which is a modification of the Gordon-Taylor model, also takes hydrogen bonding interactions between components into account. This model fitted experimental Tg data of sucrose/albumin mixtures very well. The inflection in Tg at a sucrose/albumin mass ratio of 0.5 coincides with an inflection of nOH, indicating a change in intermolecular interactions and the microcomposition of the glass. The Tg of trehalose, sucrose, and glucose determined by FTIR here is in good agreement with that of previous findings.14,15 WTC values reflect the degree of molecular packing and rearrangement during heating. The higher the WTCg value, the lower the degree of molecular packing. For carbohydrates of different sizes, WTCg and Tg increase with increasing molecular weight.15 Addition of albumin to sucrose glasses increases Tg and the average strength of hydrogen bonding (decreases WTCg). This indicates that sugars form stronger hydrogen bonds with albumin than with each other. Similar observations were made with mixtures of sucrose and poly22 L-lysine and with late embryogenesis abundant proteins.24 During storage, dried specimens can absorb water, which in turn affects the physical properties of the formulation and as a consequence stability of embedded materials. Both dried sucrose and sucrose/albumin mixtures are in a glassy state at water contents lower than ~0.04 g H2O g DW1. The change in heat capacity between the glassy and liquid states associated with the glass transition, DCp, of sucrose glasses decreases with increasing water content, whereas DCp of sucrose/albumin glasses is not affected by the water content. A greater value of DCp implies a greater change in structure with temperature increase.31 In the glassy state, the main contribution to the heat capacity comes from vibrational degrees of freedom, and the change from glassy to liquid state is associated with the unfreezing of degrees of rotational and vibrational freedom. Enthalpy relaxation of glasses gives insights into storage stability, such as aggregation32 and chemical degradation13 of compounds preserved/embedded in glasses. Fitting enthalpy relaxation data with the Kohlrausch-Williams-Watt equation allowed deriving
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parameters describing stability in glasses, namely the mean relaxation time t and the distribution of relaxation times b. The limitation of this method is that it does not take into account the time dependence of relaxation time (i.e., nonlinearity) at a constant storage temperature.33 Other approaches that can be used to fit enthalpy relaxation data taking nonlinearity into account include the Adam-Gibbs theory.34 The maximum enthalpy relaxation was lower for sucrose/albumin glasses compared to sucrose glasses, which may be attributed to the lower DCp of sucrose/albumin (see Eq. 4). At the same storage temperature, relaxation times were found to be higher for sucrose/albumin glasses compared to sucrose glasses, indicating addition of albumin increases the stability of sucrose glasses. The lower b-values of sucrose/albumin glasses indicate less homogeneity compared to sucrose glasses. A linear correlation was found when ln(t) was plotted versus Tg-Tstorage, as described before.12,35 Addition of albumin to sucrose resulted in a decrease in the slope, implicating that relaxation times in sucrose/albumin glasses increase relatively slower with increasing difference between Tg and the storage temperature. Activation energy values of enthalpy relaxation in sucrose and sucrose/albumin glasses were determined to be 332 and 236 kJ mol1, respectively. Higher activation energies have been associated with glasses being more stable during storage.35 The activation energy for sucrose is in good agreement with previous findings (21236 and 25037 kJ mol1). Conclusion A combination of FTIR spectroscopy and DSC was used here to study hydrogen bonding interactions and enthalpy relaxation phenomena of sugar and sugar/protein glasses. The sugar OH band in FTIR spectra can be used to derive the glass transition temperature, Tg, and the WTC of the OH band, whereas DSC can be used to determine Tg, the change in heat capacity associated with the glass transition, and enthalpy relaxation during storage. It is shown that the glass transition temperature and strength of hydrogen bonds of mixtures of sucrose and albumin increase with increasing percentages of albumin. The WTC of the OH band in the glassy state (WTCg) and Tg of sucrose/albumin glasses show a negative linear correlation. The Lu-Weiss equation can be used to fit Tg data of sucrose/albumin mixtures, and the inflection point that is observed at a sucrose mass fraction of 0.5 g/g coincides with an inflection of nOH denoting a change in hydrogen bonding interactions. Enthalpy relaxation of sucrose and sucrose/albumin glasses as observed in DSC thermograms after isothermal storage increases with increasing storage temperature. It is concluded that addition of albumin to sucrose increases the Tg, average strength of hydrogen bonding, heterogeneity, and the enthalpy relaxation time, making the glass more stable during storage at room temperature. Acknowledgments This work was financially supported by the German Research Foundation (DFG: Deutsche Forschungsgemeinschaft) via the Cluster of Excellence ‘From regenerative biology to reconstructive therapy’ (REBIRTH) and grant WO1735/6-1, SI1462/4-1. References 1. Crowe JH, Carpenter JF, Crowe LM. The role of vitrification during anhydrobiosis. Annu Rev Physiol. 1998;60:73-103. 2. Koster KL. Glass formation and desiccation tolerance in seeds. Plant Physiol. 1991;96:302-304. 3. Buitink J, Leprince O. Glass formation in plant anhydrobiotes: survival in the dry state. Cryobiology. 2004;48:215-228.
4. Wyatt TT, Wosten AB, Dijksterhuis J. Fungal spores for dispersion in space and time. Adv Appl Microbiol. 2013;85:43-91. 5. Wolkers WF, Walker NJ, Tablin F, Crose JH. Human platelets loaded with trehalose survive freeze-drying. Cryobiology. 2001;42:79-87. 6. Ma X, Jamil K, Macrae TH, et al. A small stress protein acts synergistically with trehalose to confer desiccation tolerance on mammalian cells. Cryobiology. 2005;51:15-28. 7. Roos Y, Karel M. Water and molecular weight effects on glass transition in amorphous carbohydrates and carbohydrate solutions. J Food Sci. 1991;56: 1676-1681. 8. Frank GA. Measurement analysis of glass transition temperature for sucrose and trehalose aqueous solutions. J Phys Chem Ref Data. 2001;36: 1279-1285. 9. Hodge IM. Physical aging in polymer glasses. Science. 1995;267:1945-1995. 10. Struik LCE. Physical Aging in Amorphous Polymers and Other Materials. Amsterdam: Elsevier; 1977. 11. Hancock BC, Shamblin SL. Molecular mobility of amorphous pharmaceuticals determined using differential scanning calorimetry. Thermochim Acta. 2001;380:95-107. 12. Mao C, Chamarthy SP, Byrn SR, Pinal R. A calorimetric method to estimate molecular mobility of amorphous solids at relatively low temperatures. Pharm Res. 2006;23:2269-2276. 13. Shamblin SL, Hancock BC, Pikal MJ. Coupling between chemical reactivity and structural relaxation in pharmaceutical glasses. Pharm Res. 2006;23: 2254-2268. 14. Wolkers WF, Oldenhof H, Alberda M, Hoekstra FA. A Fourier transform infrared microspectroscopy study of sugar glasses: application to anhydrobiotic higher plant cells. Biochim Biophys Acta. 1998;1379:83-96. 15. Wolkers WF, Oliver AE, Tablin F, Crowe JH. A Fourier-transform infrared spectroscopy study of sugar glasses. Carbohydr Res. 2004;339:1077-1085. 16. Liu Y, Bhandari B, Zhou W. Glass transition and enthalpy relaxation of amorphous food saccharides: a review. J Agric Food Chem. 2006;54: 5701-5717. 17. Hancock BC, Zografi G. The relationship between the glass transition temperature and the water content of amorphous pharmaceutical solids. Pharm Res. 1994;11:471-477. 18. Penzel E, Rieger J, Schneider HA. The glass transition temperature of random copolymers: 1. Experimental data and the Gordon-Taylor equation. Polymer. 1997;38:325-337. 19. Katkov II, Levine F. Prediction of the glass transition temperature of water solutions: comparison of different models. Cryobiology. 2004;49:62-82. 20. Lu X, Weiss RA. Relationship between the glass transition temperature and the interaction parameter of miscible binary polymer blends. Macromolecules. 1992;25:3242-3246. 21. Surewicz WK, Mantsch HH, Chapman D. Determination of protein secondary structure by Fourier transform infrared spectroscopy: a critical assessment. Biochemistry. 1993;32:389-394. 22. Wolkers WF, van Kilsdonk MG, Hoekstra FA. Dehydration-induced conformational changes of poly-L-lysine as influenced by drying rate and carbohydrates. Biochim Biophys Acta. 1998;1425:127-136. 23. Liu J, Rigsbee DR, Stotz C, Pikal MJ. Dynamics of pharmaceutical solids: the study of enthalpy relaxation by isothermal microcalorimetry. J Pharm Sci. 2002;91:1853-1862. 24. Wolkers WF, McCready S, Brandt WF, Lindsey GG, Hoekstra FA. Isolation and characterization of a D-7 LEA protein from pollen that stabilizes glasses in vitro. Biochim Biophys Acta. 2001;1544:196-206. 25. Kawai K, Hagura Y. Discontinuous and heterogeneous glass transition behavior of carbohydrate polymer-plasticizer systems. Carbohyd Polym. 2012;89:836-841. 26. Ceccorulli G, Pizzoli M, Scandola M. Composition dependence of the glass transition temperature of polymer-diluent systems: 1. Experimental evidence of a dual behavior in plasticized PVC. Polymer. 1987;28:2077-2080. 27. Kalogeras IM. Description and molecular interpretations of anomalous compositional dependences of the glass transition temperatures in binary organic mixtures. Thermochim Acta. 2010;509:135-146. 28. Braun G, Kovacs AJ. Variations in the glass transition temperature of binary systems of statistical distribution. In: Prins JA, ed. The proceedings of the conference on physics of non-crystalline solids. Amsterdam: North Holland; 1965:303. 29. Scandola M, Ceccorulli G, Pizzoli M. Composition dependence of the glass transition of polymer-diluent mixtures: 2. Two concomitant glass transition processes as a general feature of plasticized polymers. Polymer. 1987;28: 2081-2084. 30. Kwei TK. The effect of hydrogen bonding on the glass transition temperatures of polymer mixtures. J Polym Sci C: Polym Lett. 1984;22:307-313. 31. Komatsu T, Noguchi T. Heat capacity changes at the glass transition in mixedalkali tellurite glasses. J Am Ceram Soc. 1997;80:1327-1332. 32. Duddu SP, Zhang G, Dal Monte PR. The relationship between protein aggregation and molecular mobility below the glass transition temperature of lyophilized formulations containing a monoclonal antibody. Pharm Res. 1997;14:596-599. 33. Kawakami K, Pikal MJ. Calorimetric investigation of the structural relaxation of amorphous materials: evaluating validity of the methodologies. J Pharm Sci. 2005;94:948-965.
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36. Kawai K, Hagiwara T, Takai R, Suzuki T. Comparative investigation by two analytical approaches of enthalpy relaxation for glassy glucose, sucrose, maltose, and trehalose. Pharm Res. 2005;22:490-495. 37. Liu Y, Bhandari B, Zhou W. Study of glass transition and enthalpy relaxation of mixtures of amorphous sucrose and amorphous tapioca starch syrup solid by differential scanning calorimetry (DSC). J Food Eng. 2007;81: 599-610.
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Pharmaceutical Biotechnology
Hydrogen Bonding Interactions and Enthalpy Relaxation in Sugar/Protein Glasses €tte Oldenhof 2, Harald Sieme 2, Willem F. Wolkers 1, * Bulat Sydykov 1, Harrie 1 2
€t Hannover, Hannover, Germany Institute of Multiphase Processes, Leibniz Universita Clinic for HorseseUnit for Reproductive Medicine, University of Veterinary Medicine Hannover, Hannover, Germany
a r t i c l e i n f o
a b s t r a c t
Article history: Received 7 October 2016 Revised 31 October 2016 Accepted 1 November 2016
In this study, hydrogen bonding interactions and enthalpy relaxation phenomena of sugar and sugar/ protein glasses have been studied using Fourier transform infrared spectroscopy and differential scanning calorimetry. The sugar OH band in Fourier transform infrared spectra was used to derive the glass transition temperature, Tg, and the wavenumber-temperature coefficient (WTC) of the OH band. A study on mixtures of sucrose and albumin revealed that the glass transition temperature and strength of hydrogen bonds increased with increasing percentages of albumin. WTCg and Tg derived from sucrose/ albumin glasses showed a negative linear correlation. The Lu-Weiss equation was used to fit Tg data of sucrose/albumin mixtures. An inflection point was observed at a 1:1 mass ratio, which coincided with an inflection of the OH-stretching band denoting a change in hydrogen bonding interactions. Enthalpy relaxation, which is seen as an endothermic event superimposed on the glass transition in differential scanning calorimetry thermograms, increases with increasing storage temperature. Activation energies of enthalpy relaxation of sucrose and sucrose/albumin glasses were determined to be 332 and 236 kJ mol1, respectively. Addition of albumin to sucrose increases the Tg, average strength of hydrogen bonding, heterogeneity, and the enthalpy relaxation time, making the glass more stable during storage at room temperature. © 2016 American Pharmacists Association®. Published by Elsevier Inc. All rights reserved.
Keywords: amorphous calorimetry (DSC) FTIR glass proteins stability
Introduction Sugars, such as sucrose and trehalose, can protect biological materials during cryopreservation and lyophilization. Protective properties of sugars have been attributed to their ability to form hydrogen bonds and facilitate glass formation during dehydration.1,2 A glass is a highly viscous amorphous state in which molecular mobility is decreased, and damaging reactions are slowed down.2-4 Moreover, it functions as a matrix in which cellular and biomolecular structures are embedded and prevented from interacting with one another. Typical formulations used for preservation of pharmaceutical or biological materials are composed of sugars and biopolymers. Glasses composed of sugars and proteins have been implicated for dry preservation of cells.5,6 In addition to the composition, the storage temperature and relative humidity conditions are the main factors that affect molecular mobility and stability
* Correspondence to: Willem F. Wolkers (Telephone: þ49 511 762 19353; Fax: þ49 511 762 19389). E-mail address: [email protected] (W.F. Wolkers).
in the glassy state. Uptake of water has a plasticizing effect on the glass transition, which means it decreases Tg and increases molecular mobility, leading to faster degradation and crystallization processes.7 The dependence of Tg on the water content can be determined experimentally or predicted using the Gordon-Taylor equation which describes the Tg of miscible binary mixtures.7,8 Amorphous materials are in a nonequilibrium state at temperatures below Tg and have a higher specific volume and enthalpy compared to the equilibrium state. The inherent tendency of glassy materials to transition into the equilibrium state is referred to as physical aging or structural relaxation.9,10 The rate and extent of physical aging affect the longevity of preserved materials in glasses. Especially, occurrence of crystallization may cause damage to materials preserved in a glassy state. The relaxation rate in the glassy state depends on the degree of molecular mobility and the difference between the storage temperature and Tg. Differential scanning calorimetry (DSC) is one of the most widely used methods to measure glass transitions of amorphous compounds. The glass transition is evident as a change in heat flow during heating or cooling. Structural relaxation of glasses can
http://dx.doi.org/10.1016/j.xphs.2016.11.003 0022-3549/© 2016 American Pharmacists Association®. Published by Elsevier Inc. All rights reserved.
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be observed as an endothermic peak superimposed on the glass transition.11 If enthalpy relaxation is plotted versus storage duration, the rate of relaxation can be fitted using the KohlrauschWilliams-Watt equation.12 This in turn gives insights into molecular mobility in glasses,11 and stability of embedded materials.13 Fourier transform infrared (FTIR) spectroscopy also has been used to study glass transitions, particularly of carbohydrates. Glass transitions of sugars can be determined by monitoring the position of the OH-stretching vibration band (nOH) arising from sugar OH groups versus the sample temperature.14,15 When nOH is plotted versus the sample temperature, the glass transition is visible as a change in slope of nOH versus temperature above and below the glass transition. The slope is indicative of the strength of the hydrogen bonding network in the glassy or liquid state. The aim of the present study was to assess physical characteristics of sugar and sugar/protein glasses with special emphasis on factors affecting Tg, hydrogen bonding interactions, and enthalpy relaxation. It was hypothesized that adding albumin to sugar glasses increases Tg and decreases molecular mobility. Both FTIR spectroscopy and DSC were used to study Tg, molecular interactions, and enthalpy relaxation characteristics. Sugar/albumin glasses were studied at different mass ratios. The effect of water content on Tg and enthalpy relaxation at different storage temperatures was studied. Data on enthalpy relaxation during storage were fitted to derive parameters describing temperature-dependent molecular mobility in glasses. Materials and Methods Preparation of Carbohydrate Glasses Sugar glasses were prepared from glucose, sucrose (both from Carl Roth, Karlsruhe, Germany), and trehalose (Cargill, Minneapolis, MN). Sugar solutions of 20-50 mg$mL1 were prepared using distilled water. In addition, mixtures of albumin (bovine serum albumin fraction V, pH 7.0; Serva, Heidelberg, Germany) and sucrose at different sucrose/albumin mass ratios were prepared. For FTIR analyses, sugar glasses were prepared by drying 10-20 mL sugar or sugar/albumin solution on infrared transparent CaF2 windows (25 2 mm; Korth Kristalle GmbH, Altenholz, Germany). This was performed in a box purged with dry air (relative humidity less than 3%). Before measurements, residual water was removed by heating the sample (100 C for at least 3 min). The absence of water after heating was verified from the disappearance of the HOH scissoring band at ~1645 cm1. Sugar glasses for DSC analyses were prepared by freeze-drying. A volume of 1 mL of sugar or sugar/albumin solution was added in freeze-drying vials (2R injection vials; Landgraf Laborsysteme, Langenhagen, Germany), and vials were placed on the temperaturecontrolled shelves of a lyophilizer (Virtis Advantage Plus Benchtop freeze dryer; SP Scientific, Warminster, PA). Samples were cooled from 20 C down to 30 C at 1 C min1 and kept at 30 C for 1 h. Primary drying was performed at a temperature of 30 C and a pressure of 60 mTorr, for 15 h. Then, the shelf temperature was increased to 40 C, at 0.1 C min1 while maintaining a pressure of 60 mTorr. Samples were maintained at 40 C for 1 h, after which secondary drying was performed at a temperature of 20 C and pressure of 10 mTorr for 6 h. After freeze-drying, freeze-dried material was directly used for DSC analysis or processed for storage. Samples with different water contents were obtained by placing pans with freeze-dried material in containers with different relative humidity before sealing them. A relative humidity of 35% and 75% was obtained using saturated salt solutions of CaCl2 and NaCl, respectively.
Fourier Transform Infrared Spectroscopy Studies Infrared spectra were recorded using a Perkin-Elmer 100 FTIR spectrometer (Perkin Elmer, Norwalk, CT), equipped with a triglycine sulfate detector, a temperature-controlled sample holder connected to a heating device (Harrick Scientific Products, Pleasantville, NY), and a Linkam pump system for using liquid nitrogen as a coolant (Linkam Scientific Instruments, Tadworth, Surrey, UK). The optical bench was continuously purged with dry air from an FTIR purge gas generator (Whatman, Clifton, NJ). Spectra acquisition parameters were as follows: 4 cm1 resolution, 4 co-added interferograms, and 4000-900 cm1 wavenumber range. Sugar glasses prepared on a CaF2 window, as described above, were covered with a second window while having a Teflon spacer between them to avoid sticking. This was mounted in the sample holder, residual water was removed as described above, and the sample temperature was decreased to 30 C, at 1 C min1. Then, the temperature was increased to temperatures up to 180 C, at 1 C min1, while acquiring spectra every 30-60 s. A T-type thermocouple (Fluke, Everett, WA) was used to monitor the sample temperature. In addition to using Perkin-Elmer software, spectral analysis and display were carried out using Omnic (ThermoElectron Corporation, Waltham, MA) as well as Wolfram Mathematica software (Wolfram Research, Champaign, IL). Melting of sugar glasses was monitored by following the position of nOH around 3300 cm1 versus the sample temperature, as previously described in detail.14,15 In short, the 3600-3000 cm1 spectral region was selected and normalized, after which the band position was calculated as the average of the spectral positions at 80% of the full peak height. Plots were constructed in which nOH was plotted as a function of the sample temperature, and linear regression lines in both the liquid and glassy state regions of the plot were added. The glass transition temperature (Tg) was determined as the intersection point of these 2 regression lines, whereas wavenumber-temperature coefficients (WTCg and WTCl) refer to the slopes of the regression lines. Differential Scanning Calorimetry DSC measurements were taken using a Netzsch DSC 204F1 Phoenix instrument (Netzsch-Geraetebau GmbH, Selb, Germany). Calibration was performed using adamantane, bismuth, indium, zinc, selenium, and cesium chloride, according to the instructions provided by the manufacturer. Approximately 10 mg of freezedried sample, prepared as described above, was added into a 25-mL aluminum pan. After sealing pans hermetically, the fresh sample weight was determined using a microbalance. Sealed pans with samples were either directly used for DSC analysis or stored for different durations (up to ~1 week) at different temperatures (1 C50 C) for analyses of glass characteristics during aging. With DSC measurements, an empty pan was used as a reference sample. In order to study glass transitions, samples were cooled to 50 C with 10 C min1 and held there for 5 min, after which they were heated to 150 C at 10 C min1, while monitoring the heat flow. Thermal events were determined from the obtained thermograms, using Netzsch software. Dry weights of samples were determined after DSC analyses, after perforating the pans and overnight incubation in an oven set at 80 C. Water contents were determined by comparing the fresh and dry weight:
WC ¼
½ðFW þ PÞ P ½ðDW þ PÞ P ½ðDW þ PÞ P
(1)
FW and DW represent the fresh and dry weights of the sample, respectively, P is the weight of the pan (all in grams), and WC is the water content (in g H2O per g DW).
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The glass transition temperature (Tg) was determined as the midpoint of the temperature range, over which the change in specific heat coinciding with the transition from glassy to liquid state occurred, whereas DCp is the change in heat flow at Tg divided by the heating rate. Enthalpy relaxation (or enthalpy recovery) was observed as an endothermic event occurring superimposed on top of the glass transition and quantified as the area under the endothermic peak associated with Tg, according to Liu et al.16 as described below. Glass Transition Models of Binary Mixtures Different models were tested for predicting the dependence of the glass transition temperature on the composition of binary mixtures, including the Gordon-Taylor and Lu-Weiss models. The Gordon-Taylor model was originally derived for polymer blends and copolymers assuming that the volumes of the 2 components are additive and there are no specific interactions between the components17:
x1 Tg;1 þ kð1 x1 ÞTg;2 Tg ¼ x1 þ kð1 x1 Þ
(2)
Here x1 and Tg,1 are the mass fraction and the glass transition temperature (in Kelvin) of the first component, whereas Tg,2 is of the second component. The parameter k is defined as r1Da2/r2Da1, where Da is the change in coefficient of thermal expansion at Tg, and r1 and r2 represent the density of the first and second component. Actually, k can be used as a parameter to describe interactions between components in a binary mixture,18 such as hydrogen bond interactions in water-carbohydrate mixtures.19 The Lu-Weiss model describes the glass transition temperature behavior for miscible binary polymer blends taking into account interactions (e.g., ionic interactions, hydrogen bonding) between components20:
Tg ¼
x1 Tg;1 þ k$ð1 x1 ÞTg;2 þ Ax1 ð1 x1 Þð1 þ dð1 x1 ÞÞ x1 þ k$ð1 x1 Þ
(3)
In addition to the parameters described for the Gordon-Taylor equation, k, A, and d are used as fitting parameters. k represents the ratio of the change of heat capacities at the Tg of the second versus the first component, A is a parameter describing the interactions, and d contains information about the linear dependency of k and densities and molecular weight ratios of the 2 components.
3
Here DCp (in J$g1$K1) is the change in heat capacity at Tg, and T (in Kelvin) represents the storage temperature. Enthalpy relaxation during storage then can be fitted using:
( DHrelaxation ðtÞ ¼ DH∞
"
b #) t 1 exp t
(5)
This requires 2 fitting parameters, namely t which is the mean relaxation time (in seconds) and b (which is 0 and 1) representing a nonexponential parameter describing the distribution of relaxation times. b is equal to 1 in case of a single relaxation time whereas smaller values for b are indicative of a wider distribution of t. Arrhenius plots of relaxation time versus storage temperature were fitted using:
ln t ¼
Ea 1000 þ lnt0 T R
(6)
where R is the gas constant (8.314 J mol1 K1), the mean relaxation time t is given in seconds, and storage temperature T in Kelvin. The activation energy Ea (in kJ mol1) follows from the slope of the linear plot, whereas t0 can be calculated from the intersection point. Results Infrared Spectra and Temperature Dependence of nOH In Figure 1, typical infrared spectra of sugar glasses, prepared from glucose, sucrose, and trehalose, are shown. In addition, spectra of crystalline sucrose and albumin are shown. Residual water is evident as a band around 1650 cm1 arising from HOHscissoring vibrations. OH-stretching vibrations resulting from sugars can be found in the 3600-3000 cm1 region. It can be seen that absorbance bands in amorphous sucrose are broader compared to crystalline sucrose. This is explained by the more variable lengths and orientations of hydrogen bonds as present in a glass. Also, the peak position of the OH band of crystalline sucrose is shifted to a lower wavenumber, indicating stronger hydrogen bonds compared to those in the amorphous state. The 3000-2800 cm1 region contains CH-stretching vibrations arising from the
Enthalpy Relaxation DSC pans with sucrose and sucrose/albumin (1:1 mass ratio) glasses were stored for different durations at different temperatures as described above. Enthalpy relaxation values were derived from DSC thermograms and plotted versus the storage time. Enthalpy relaxation of stored sucrose/albumin glasses was determined by subtracting the thermogram of a nonaged sample. In case of sucrose glasses, the thermogram was extrapolated from above Tg to lower temperatures without the endothermic glass relaxation peak, and this curve was subtracted from the original thermogram exhibiting enthalpy relaxation.11 To derive parameters describing molecular mobility in a glass, the dependence of the enthalpy relaxation (DHrelaxation in J g1) on the storage duration (t in seconds) was fitted using the KohlrauschWilliams-Watt equation. First, the maximum possible enthalpy recovery DH∞ at a given storage temperature is described by:
DH∞ ¼ DCp Tg T :
(4)
Figure 1. Infrared absorption spectra of sugar glasses, albumin (BSA), and crystalline sucrose. nOH is visible around 3300 cm1, and the CH2-stretching region is from 3000-2800 cm1. The amide-I and amide-II bands of BSA are visible at 1650 and 1550 cm1, respectively.
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carbohydrate methyl groups. The 1500-1000 cm1 region is referred to as the “fingerprint” region which, in case of sugars, contains absorbance bands arising from CH-deformation, C¼O stretching, and OH-bending vibrations. The spectrum of albumin displays 2 main bands at around 1650 and 1550 cm1, which can be attributed to the amide I and amide II band, respectively.21 Albumin also has an OH band arising from intrinsic OH groups. However, the protein OH band is relatively weak compared to that of the sugars, because sugars have a relatively high number of OH groups.22 The temperature dependence of nOH position (~3300 cm1) was studied to determine glassy behavior and glass transition temperatures (Fig. 2a). With increasing sample temperature, molecules will have more thermal energy, which leads to a reduced level of hydrogen bonding and a shift of the nOH to a higher wavenumber. At the glass transition temperature, there is a change of slope in the nOH wavenumber position versus temperature plot. The Tg was derived from the intersections of linear regression lines in the glassy and liquid regions, below and above Tg, and was determined to be 108 ± 3 C for trehalose, 58 ± 1 C for sucrose, and 30 ± 2 C for glucose (Fig. 2b). The slopes of the regression lines (WTC) are a measure of the strength of the hydrogen bonding network; the higher the value of WTC, the weaker the hydrogen bonds. In the glassy state, the WTC value is lower (0.205-0.290 C cm1) as compared to the liquid state (0.518-0.548 C cm1), indicating stronger hydrogen bonds. In the liquid state, no large differences were found between WTC values of the studied materials. In the glassy state, however, the WTC for glucose was lower as compared to values determined for sucrose and trehalose. This most likely is due to the smaller size of glucose compared to that of disaccharides.
hydrogen bonds (i.e., decreased WTCg). For sucrose/albumin mixtures, with relative sucrose content up to ~0.5 g/g, a negative linear correlation was found when WTCg was plotted versus Tg (Fig. 4a). This is different from the positive linear correlation that was found when plotting data obtained for glasses made from pure sugars with different molecular weights.15 It should be noted that albumin also has an OH band arising from intrinsic OH groups (Fig. 1). However, in sucrose/albumin mixtures, the OH band is dominated by the OH band of sugars. At higher albumin contents, however, the band may also comprise a contribution of the albumin OH band. The band position of the protein OH groups hardly changes with temperature and therefore the glass transition of albumin alone cannot be measured by this FTIR method, because in contrast to sugar OH groups, protein OH groups do not directly interact with the glassy matrix. The glass transition can only be detected if sugars are present and can be determined at sucrose contents as low as 0.1 g/g. When Tg was plotted versus the relative sucrose content in the sucrose/albumin mixture, an inflection point is seen at relative sucrose contents of 0.4-0.6 g/g, below which a linear correlation can be seen (Fig. 4b). The Tg of pure albumin was estimated to be 178 C via extrapolation of data in the linear region (below 0.4) toward 0. The Gordon-Taylor equation, which assumes absence of specific interactions in a mixture, could not be used to fit the relation between Tg and the relative sucrose content in sucrose/ albumin mixtures. This is likely due to hydrogen bonding interactions between the components, as evident from plots of nOH versus the relative sucrose content (Fig. 4b). Data were successfully fitted with the Lu-Weiss equation, which takes into account interactions between components in a mixture (with k ¼ 0.162, A ¼ 149, d ¼ 3.96).
Glass Transition Temperatures and WTC of Sucrose/Albumin Glasses Glasses prepared using different sucrose/albumin mass ratios were subjected to FTIR analysis, and nOH was monitored versus the sample temperature in order to derive Tg and WTC values (Fig. 3a). In Figure 3b, it can be seen that an increased relative percentage of albumin (i.e., lower sucrose/albumin mass ratio value) resulted in an increase in the glass transition temperature and strength of
Effect of Water Content on Tg and DCp of Sucrose and Sucrose/ Albumin Glasses DSC was used to analyze effects of the sample water content on characteristics of both sucrose and sucrose/albumin (1:1 mass ratio) glasses. Tg was determined as the onset temperature at which the change in specific heat coinciding with the transition
Figure 2. FTIR was used for studying temperature-dependent glass-to-liquid transitions in sugar glasses. This was performed for the disaccharides trehalose (white circles, bars without filling) and sucrose (grey circles, bars with diagonals), as well as monosaccharide glucose (black circles, bars with diamonds). Infrared spectra were collected during heating, and the position of nOH was determined and plotted versus the sample temperature (a). The glass transition temperature (Tg) was determined as the intersection point of the regression lines in the liquid state and in the glassy state (b), whereas WTC represent the slopes of the regression lines in both states (c; WTCg and WTCl). Mean values ± standard deviations are presented, determined from 6 independent experiments.
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5
Figure 3. FTIR analyses of glasses prepared using different mass ratios of sucrose (SUC) and albumin (BSA). In panel a, plots are shown on nOH versus the sample temperature; for SUC/BSA glasses prepared at mass ratios (w/w) of 9:1 (white circles), 6:4 (grey circles), and 2:8 (black circles). In panel b, plots are shown on the Tg (white squares) as well as the WTCg (black squares) versus the sucrose/albumin mass ratio.
from glassy to liquid state occurred, whereas DCp was defined as the change in heat flow at Tg divided by the heating rate (Fig. 5a). It was found that Tg decreases with increasing water content, both for sucrose and sucrose/albumin glasses, which can be attributed to the plasticizing effect of water. At water contents lower as 0.05 g water per gram dry weight, the Tg of sucrose is higher as compared to sucrose/albumin glasses. At higher water contents, a glass transition of 27 C (>0.05 g H2O g DW1) was determined for sucrose glasses, which did not further decrease. This refers to Tg’, which is the Tg of the maximally freeze-concentrated amorphous phase. For sucrose/albumin glasses, Tg values continued to decrease. The DCp values associated with the glass transition were found to be greater for sucrose glasses compared to sucrose/ albumin mixtures at water contents below 0.05 g H2O g DW1. For sucrose glasses, however, DCp decreased with below 0.05 g H2O g DW1, whereas DCp of sucrose/albumin glasses remained constant as a function of the water content. It should be noted that the heat flow was not calibrated for Cp measurements using sapphire as a reference standard. The DCp values were approximated directly from the heat flow, which causes inaccuracies in the reported values.
Enthalpy Relaxation in Sucrose and Sucrose/Albumin Glasses In sugar glasses, there is molecular mobility and enthalpy relaxation, which is seen as an endothermic event superimposed on the glass transition in DSC thermograms (Fig. 5a). For sucrose and sucrose/albumin glasses (1:1mass ratio), DHrelaxation was determined as the area of this peak and monitored during storage at different temperatures. Representative DSC thermograms are shown in Figures 6a and 6b, and DHrelaxation versus storage duration is plotted in Figures 6c and 6d. At a given time point, enthalpy relaxation and thus glass molecular mobility were determined to be higher with increasing storage temperature, with larger effects for sucrose glasses as compared to sucrose/ albumin glasses. Data were fitted using the Kohlrausch-WilliamsWatt equation, to derive the maximum possible enthalpy recovery DH∞, the mean relaxation time t, and parameter b (stretch power) describing the distribution of relaxation times. Data are summarized in Table 1. DH∞ was found to increase with decreasing storage temperature, and DH∞ was found to be lower for sucrose/albumin glasses compared to sucrose glasses. Values for t increased with decreasing storage temperature for both
Figure 4. In panel a, a correlation plot is shown on WTCg values in the glassy state versus the Tg, determined using FTIR, for glasses prepared from sucrose/albumin mixtures (black squares; 1:10-9:10 mass ratios). In addition, data are presented for glasses made from various oligo- and polysaccharides (white squares: glucose, octulose, sucrose, umbelliferose, trehalose, raffinose, glucan, and dextran-T40, adapted from the study by Wolkers et al.15). In panel b, the Tg (black circles) and the nOH peak position at 25 C (white circles) are plotted versus the relative sucrose content in sucrose/albumin glasses. The dependency of Tg on the relative sucrose content was fitted using the Lu-Weiss equation.
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B. Sydykov et al. / Journal of Pharmaceutical Sciences xxx (2016) 1-9
Figure 5. DSC was used to study the glassy behavior of sucrose (white squares) and sucrose/albumin (1:1 mass ratio; black squares) glasses of different water contents. In panel a, representative DSC thermograms (heating scans) are presented to illustrate how Tg, DCp, and DHrelaxation were derived. In panels b and c, Tg and DCp are plotted versus the sample water content. Exothermic events are oriented downward.
Figure 6. DSC was used to study enthalpy relaxation behavior of sucrose (a and c) and sucrose/albumin (1:1 mass ratio; b and d) glasses during storage at different temperatures. In panels a and b, representative thermograms are shown to illustrate differences. Exothermic events are oriented downward. In panels c and d, DHrelaxation is plotted as a function of the storage duration, for sucrose (white squares: 1 C, light grey squares: 22 C, dark grey squares: 30 C, black squares: 38 C) and sucrose/albumin glasses (white squares: 1 C, light grey squares: 22 C, dark grey squares: 37 C, black squares: 52 C), respectively. Data were fitted using the Kohlrausch-Williams-Watt equation, for deriving parameters describing molecular mobility in glasses.
B. Sydykov et al. / Journal of Pharmaceutical Sciences xxx (2016) 1-9 Table 1 Parameters Describing Molecular Mobility in Sucrose and Sucrose/Albumin Glasses (1:1 Mass Ratio) Stored at Different Temperatures, as Derived From Fitting the Enthalpy Relaxation Data Obtained From DSC Measurements Versus Storage Duration, Using the Kohlrausch-Williams-Watt Model Compound
Tstorage ( C)
DH∞ (J kg1)
t (d)
b
Sucrose
38 30 22 1 52 38 22 1
11.34 18.70 26.06 45.37 5.77 10.97 16.92 24.73
1.62 43 99 52614200 0.92 49 495860 5089760
0.43 0.31 0.44 0.23 0.40 0.26 0.15 0.20
Sucrose/albumin
Compound
Tstorage ( C)
Tg ( C)
t (d)
b
Trehalose Sucrose
40 50
113 69.4
5565186 12
0.188 0.199
Parameters include the following: the maximum possible enthalpy recovery
DHrelaxation (in J kg1), the mean relaxation time t (in d), and parameter b describing the distribution of relaxation times (equals 1 in case of a single relaxation, where lower values are indicative of a wider distribution). For comparison, data of freeze-dried sucrose and trehalose from Liu et al.23 are included (last 2 rows).
sucrose and sucrose/albumin glasses. Values for b were lower for sucrose/albumin glasses, indicating a wider distribution of the relaxation time (i.e., less homogenous glass) as compared to sucrose glasses. In order to visualize differences between temperaturedependent glass relaxation parameters in sucrose and sucrose/ albumin glasses, plots were constructed in which the natural logarithm of t was plotted versus the difference between Tg and Tstorage (Fig. 7). This gives a linear correlation, with sucrose glasses exhibiting higher values for ln(t) and a steeper slope compared to sucrose/albumin glasses. An Arrhenius plot (inset in Fig. 7) was used to derive activation energies. Activation energies of enthalpy relaxation for sucrose and sucrose/albumin glasses were determined to be 332 and 236 kJ mol1, respectively.
Figure 7. Parameters describing molecular mobility in glasses were determined by fitting DSC data on enthalpy relaxation versus storage, both for sucrose (white squares) and for sucrose/albumin glasses (1:1 mass ratio; black squares). The natural logarithm of the relaxation time t is plotted versus the temperature stored above Tg. In the inset figure, data are presented in Arrhenius plots for deriving activation energies.
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Discussion FTIR and DSC were used to study physical properties of sucrose and sucrose/albumin glasses. Glasses composed of sugars and proteins have been implicated for dry preservation of cells.5,6 Albumin has been used as protein in these cases. Biological glasses in anhydrobiotic organisms also comprise of a mixture of sugars and proteins such as late embryogenesis abundant proteins that protect during drying. Among other protective functions, these proteins form a glass together with sugars and increase the Tg and molecular packing density of the cytoplasmic glassy matrix in desiccation-tolerant cells.24 The Tg of anhydrous sucrose/albumin glasses is dependent on the mass fraction of sucrose, and a plot of Tg versus sucrose content shows a sigmoidal-shaped curve (Fig. 4b). A similar sigmoidalshaped curve has been observed for maltose/dextrin and glucose/ dextrin glasses25 as well as for mixtures of polyvinyl chloride and phthalates.26 According to Kawai and Hagura,25 binary systems are composed of different regions with different microcompositions. At high relative sucrose content, sucrose predominantly interacts with other sucrose molecules, whereas at sucrose contents below 0.5 g/g, sugars form hydrogen bonds with albumin. Different models can be used to fit Tg data of binary mixtures.27 The Gordon-Taylor equation/model is most commonly used. In cases where there are multiple regions with different microcompositions, such as in binary mixtures, an inflection point is often observed above and below a critical composition which can be described using the models of Braun and Covacs28 and Scandola et al.29 The Kwei equation30 takes into account hydrogen bond interactions between components. The Lu-Weiss equation,20 which is a modification of the Gordon-Taylor model, also takes hydrogen bonding interactions between components into account. This model fitted experimental Tg data of sucrose/albumin mixtures very well. The inflection in Tg at a sucrose/albumin mass ratio of 0.5 coincides with an inflection of nOH, indicating a change in intermolecular interactions and the microcomposition of the glass. The Tg of trehalose, sucrose, and glucose determined by FTIR here is in good agreement with that of previous findings.14,15 WTC values reflect the degree of molecular packing and rearrangement during heating. The higher the WTCg value, the lower the degree of molecular packing. For carbohydrates of different sizes, WTCg and Tg increase with increasing molecular weight.15 Addition of albumin to sucrose glasses increases Tg and the average strength of hydrogen bonding (decreases WTCg). This indicates that sugars form stronger hydrogen bonds with albumin than with each other. Similar observations were made with mixtures of sucrose and poly22 L-lysine and with late embryogenesis abundant proteins.24 During storage, dried specimens can absorb water, which in turn affects the physical properties of the formulation and as a consequence stability of embedded materials. Both dried sucrose and sucrose/albumin mixtures are in a glassy state at water contents lower than ~0.04 g H2O g DW1. The change in heat capacity between the glassy and liquid states associated with the glass transition, DCp, of sucrose glasses decreases with increasing water content, whereas DCp of sucrose/albumin glasses is not affected by the water content. A greater value of DCp implies a greater change in structure with temperature increase.31 In the glassy state, the main contribution to the heat capacity comes from vibrational degrees of freedom, and the change from glassy to liquid state is associated with the unfreezing of degrees of rotational and vibrational freedom. Enthalpy relaxation of glasses gives insights into storage stability, such as aggregation32 and chemical degradation13 of compounds preserved/embedded in glasses. Fitting enthalpy relaxation data with the Kohlrausch-Williams-Watt equation allowed deriving
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parameters describing stability in glasses, namely the mean relaxation time t and the distribution of relaxation times b. The limitation of this method is that it does not take into account the time dependence of relaxation time (i.e., nonlinearity) at a constant storage temperature.33 Other approaches that can be used to fit enthalpy relaxation data taking nonlinearity into account include the Adam-Gibbs theory.34 The maximum enthalpy relaxation was lower for sucrose/albumin glasses compared to sucrose glasses, which may be attributed to the lower DCp of sucrose/albumin (see Eq. 4). At the same storage temperature, relaxation times were found to be higher for sucrose/albumin glasses compared to sucrose glasses, indicating addition of albumin increases the stability of sucrose glasses. The lower b-values of sucrose/albumin glasses indicate less homogeneity compared to sucrose glasses. A linear correlation was found when ln(t) was plotted versus Tg-Tstorage, as described before.12,35 Addition of albumin to sucrose resulted in a decrease in the slope, implicating that relaxation times in sucrose/albumin glasses increase relatively slower with increasing difference between Tg and the storage temperature. Activation energy values of enthalpy relaxation in sucrose and sucrose/albumin glasses were determined to be 332 and 236 kJ mol1, respectively. Higher activation energies have been associated with glasses being more stable during storage.35 The activation energy for sucrose is in good agreement with previous findings (21236 and 25037 kJ mol1). Conclusion A combination of FTIR spectroscopy and DSC was used here to study hydrogen bonding interactions and enthalpy relaxation phenomena of sugar and sugar/protein glasses. The sugar OH band in FTIR spectra can be used to derive the glass transition temperature, Tg, and the WTC of the OH band, whereas DSC can be used to determine Tg, the change in heat capacity associated with the glass transition, and enthalpy relaxation during storage. It is shown that the glass transition temperature and strength of hydrogen bonds of mixtures of sucrose and albumin increase with increasing percentages of albumin. The WTC of the OH band in the glassy state (WTCg) and Tg of sucrose/albumin glasses show a negative linear correlation. The Lu-Weiss equation can be used to fit Tg data of sucrose/albumin mixtures, and the inflection point that is observed at a sucrose mass fraction of 0.5 g/g coincides with an inflection of nOH denoting a change in hydrogen bonding interactions. Enthalpy relaxation of sucrose and sucrose/albumin glasses as observed in DSC thermograms after isothermal storage increases with increasing storage temperature. It is concluded that addition of albumin to sucrose increases the Tg, average strength of hydrogen bonding, heterogeneity, and the enthalpy relaxation time, making the glass more stable during storage at room temperature. Acknowledgments This work was financially supported by the German Research Foundation (DFG: Deutsche Forschungsgemeinschaft) via the Cluster of Excellence ‘From regenerative biology to reconstructive therapy’ (REBIRTH) and grant WO1735/6-1, SI1462/4-1. References 1. Crowe JH, Carpenter JF, Crowe LM. The role of vitrification during anhydrobiosis. Annu Rev Physiol. 1998;60:73-103. 2. Koster KL. Glass formation and desiccation tolerance in seeds. Plant Physiol. 1991;96:302-304. 3. Buitink J, Leprince O. Glass formation in plant anhydrobiotes: survival in the dry state. Cryobiology. 2004;48:215-228.
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