Mechanisms and models of dehydration and slow freezing damage to cell membranes

University of Minnesota Ph.D. dissertation. October 2010. Major: Mechanical Engineering. Advisor: Alptekin Aksan. 1 computer file (PDF) xviii, 199 pages.Cell preservation is accomplished primarily by two methods: cryopreservation and dehydration, with the former being the standard technique used. In order to optimize and develop cell preservation protocols for cells that are difficult to preserve or whose end application is incompatible with current cell preservation protocls and to advance preservation by dehydration, a better understanding of the freeze- and dehydration-induced changes to the cell membrane is required. Despite a large body of literature on the topic, the mechanisms of damage to cells during slow freezing and dehydration are still ambiguous. The objective of this study is to investigate the mechanisms of damage to the cell membrane during slow freezing and dehydration and expand our outlook beyond the cell membrane to its underlying support, the cytoskeleton. In this study, we used several model systems to investigate slow freezing and dehydration. We used a liposome model to gather basic information on changes that can occur to a simple membrane system during freezing. This study revealed that eutectic formation was capable of dehydrating the membrane at low temperatures which may be contribute to alteration of the post-thaw membrane structure. We used a bacteria model to investigate the role of the phase transition and immediate versus slow osmotic stress on post-rehydration viability. This study revealed that going through a lyotropic membrane phase transition was detrimental to post-rehydration viability. This study also demonstrated that a rapidly applied osmotic stress was more detrimental to the structure/ organization of the membrane than gradual osmotic stress. We then subjected a model mammalian cell to both hyperosmotic stress and freeze-thaw and investigated both the membrane and cytoskeletal responses. Osmotic stress experiments suggested that alterations in membrane structure (i.e., surface defects and lipid dissolution) were directly dependent on the change in the chemical potential of water. These experiments also suggest that cell shrinkage and the resulting formation of membrane protrusions negatively affect viability upon return to isotonic conditions. It was found that membrane morphology in the dehydrated state and post-hyperosmotic viability was dependent on the stiffness of the cytoskeleton. Freeze/ thaw experiments suggested that ice-cell interaction decreases post-thaw viability. However, similar to osmotic stress experiments, cell shrinkage and cytoskeletal stiffness negatively impact post-thaw viability. We suggest the resulting membrane morphology due to cell shrinkage is also responsible for damage during freeze/ thaw. The various mechanisms discovered and the models proposed can be used in developing new protocols for cell preservation and for cell destruction (e.g. cryosurgery)

SBC2008-192461 DESICCATION OF GEOBACTER SULFURREDUCENS: MEMBRANE RESPONSE TO OSMOTIC STRESS

INTRODUCTION Geobacter sulfurreducens is a δ-proteobacterium capable of electron transfer to extracellular compounds and surfaces Technological applications would benefit tremendously if methods to stabilize, preserve and store G. sulfurreducens at noncryogenic temperatures could be devised. As such, stabilization by desiccation provides an attractive alternative. During desiccation, the structure of the cell membrane changes due to lyotropic phase transitions, phase separation, fusion, etc. It has been proposed that the kinetics and temperatures of dehydration Therefore, both the development of thin films containing encapsulated bacteria and the stabilization of bacteria at non-cryogenic temperatures requires consideration of the membrane response. In this study we focused on quantifying the response of G. sulfurreducens through controlled desiccation experiments, and employed FTIR spectroscopy to investigate the structural and phase transitions of membranes. MATERIALS AND METHODS G. sulfurreducens strain PCA (ATCC #51573) was sub-cultured in our laboratory at 30 °C using an anaerobic medium. For these experiments 10 ml aliquots of cell suspension were anaerobcially centrifuged (5 min, 5,000 x g) and resuspended in either 20 μl of fresh growth medium, or growth medium containing sucrose. Samples were either exposed to osmotic shock or gradually dried at a drying temperature (T d ) that would either allow a lyotropic phase change (T d =30ºC) or prevent it (T d =5ºC). Viability experiments were performed in an anaerobic glove bag (H 2 :CO 2 :N 2 5:20:75) after the desiccated cells were re-hydrated. A fluorescence-based viability assay (Baclight L7012; Molecular Probes, Portland, OR) based on membrane permeability was used to determine cell viability. Viability changes were verified by measuring colony forming units on solid medium. Temperature ramp Fourier Transform Infrared Spectroscopy (FTIR) measurements were performed to monitor the changes in the phase transition behavior of the cell membrane. Membrane fluidity was quantified by measuring the change in the location of the νCH 2 symmetric stretching band maximum, which was located approximately at 2850 cm −1 . Higher νCH 2 wavenumbers indicated higher membrane fluidity. Temperature dependent shift in the νCH 2 peak location was used to determine membrane phase transition temperature (T m ) using first derivative analysis. Cooperativity of the membrane phase transition was determined by measuring the slope of the νCH 2 change at T m . RESULTS To investigate the effects of osmotic shock on G. sulfurreducens, cells were re-suspended in growth medium solutions containing up to 27% w/w sucrose. Lower concentrations of sucrose (6.75% w/w) neither significantly affected the cooperativity of the membrane phase transition nor the viability To determine the effects of gradually increased osmotic stress, cells were suspended in growth medium solutions that contained 0% or 13.5% w/w sucrose, and dried at T d =30 ºC for up to 1 hour. With increased drying time, there was an increase in T m . Cells dried in the presence of sucrose had higher T m values as well as lower viabilities. The increase in T m values with drying time was due to the reduction in water activity, which decreased viability. To investigate the effect of lyotropic phase transition, cells were dried at T d =5ºC and T d =30ºC for 45 minutes in growth medium that contained 0% and 6.75% w/w sucrose. The environmental relative humidity was adjusted so that cells are exposed to the same environmental water activity at both temperatures. Cells dried without sucrose at 30ºC had decreased phase transition cooperativity as compared to the control (undried) sample DISCUSSION The large increase in the fluidity in the gel phase and the decrease in the phase transition cooperativity for G. sulfurreducens in growth medium + 27% w/w sucrose ( We conclude that by avoiding lyotropic phase changes during drying, it is possible to lessen deleterious alterations to the membrane and obtain a higher percentage of viable cells upon desiccation. ACKNOWLEDGEMENT

The Role of Polymer Concentration on the Molecular Mobility and Physical Stability of Nifedipine Solid Dispersions

We investigated the influence of polymer concentration (2.5–20% w/w) on the molecular mobility and the physical stability in solid dispersions of nifedipine (NIF) with polyvinylpyrrolidone (PVP). With an increase in polymer concentration, the α-relaxation times measured by broadband dielectric spectroscopy were longer, which reflects a decrease in molecular mobility. In the supercooled state, at a given temperature (between 55 and 75 °C), the relaxation time increased linearly as a function of polymer concentration (2.5–20% w/w). The temperature dependence of the relaxation time indicated that the fragility of the dispersion, and by extension the mechanism by which the polymer influences the relaxation time, was independent of polymer concentration. The time for NIF crystallization also increased as a function of polymer concentration. Therefore, by using molecular mobility as a predictor, a model was built to predict NIF crystallization from the dispersions in the supercooled state. The predicted crystallization times were in excellent agreement with the experimental data

Quantification, Mechanism, and Mitigation of Active Ingredient Phase Transformation in Tablets

Model tablet formulations containing thiamine hydrochloride [as a nonstoichiometric hydrate (NSH)] and dicalcium phosphate dihydrate (DCPD) were prepared. In intact tablets, the water released by dehydration of DCPD mediated the transition of NSH to thiamine hydrochloride hemihydrate (HH). The use of an X-ray microdiffractometer with an area detector enabled us to rapidly and simultaneously monitor both the phase transformations. The spatial information, gained by monitoring the tablet from the surface to the core (depth profiling), revealed that both DCPD dehydration and HH formation progressed from the surface to the tablet core as a function of storage time. Film coating of the tablets with ethyl cellulose caused a decrease in both the reaction rates. There was a pronounced lag time, but once initiated, the transformations occurred simultaneously throughout the tablet. Thus the difference in the phase transformation behavior between the uncoated and the coated tablets could not have been discerned without the depth profiling. Incorporation of hydrophilic colloidal silica as a formulation component further slowed down the transformations. By acting as a water scavenger it maintained a very “dry” environment in the tablet matrix. Finally, by coating the NSH particles with hydrophobic colloidal silica, the formation of HH was further substantially decelerated. The microdiffractometric technique not only enabled direct analyses of tablets but also provided the critical spatial information. This helped in the selection of excipients with appropriate functionality to prevent the <i>in situ</i> phase transformations

The Benefit of Dividing an Indirect Thermal Storage into Two Compartments: Discharge Experiments

Experiments are presented to demonstrate the benefits of dividing an indirect thermal storage into two compartments. The transient discharge experiments were conducted in an undivided and equally divided 126 l rectangular storage vessel, which has a height to depth aspect ratio of 9.3:1 and is inclined at 30° to the horizontal. A 240-tube copper heat exchanger with a total surface area of 2.38 m2 was immersed in the storage fluid. For the divided storage, the heat exchanger flow path was in series through the two compartments. Water flow rate through the heat exchanger was varied from 0.05 to 0.15 kg/s to demonstrate the effect of varying the number of transfer units (NTU) from 2.2 to 7 on the relative performance of undivided and divided storage vessels. Reported measurements include transient storage temperature distribution, heat exchanger outlet temperature, delivered energy, and exergy of the divided and undivided storage. The divided storage provides higher energy delivery rates and higher heat exchanger outlet temperatures during most of the discharge. The magnitude of these benefits depends on NTU and the extent of discharge. For a flow rate of 0.05 kg/s, corresponding to a nominal NTU of 7, the divided storage delivers a maximum of 11% more energy than the undivided storage when 100 l of hot water or 55% of the stored energy has been delivered. For a flow rate of 0.15 kg/s, corresponding to a nominal NTU of 2.5, the divided storage delivers a maximum of 5% more energy at the same level of discharge. Data agree with first and second law analyses of a storage system comprised of two tanks in series

The Role of Drug–Polymer Hydrogen Bonding Interactions on the Molecular Mobility and Physical Stability of Nifedipine Solid Dispersions

We investigated the influence of drug–polymer hydrogen bonding interactions on molecular mobility and the physical stability in solid dispersions of nifedipine with each of the polymers polyvinylpyrrolidone (PVP), hydroxypropylmethyl cellulose (HPMCAS), and poly­(acrylic acid) (PAA). The drug–polymer interactions were monitored by FT-IR spectroscopy, the molecular mobility was characterized using broadband dielectric spectroscopy, and the crystallization kinetics was evaluated by powder X-ray diffractometry. The strength of drug–polymer hydrogen bonding, the structural relaxation time, and the crystallization kinetics were rank ordered as PVP > HPMCAS > PAA. At a fixed polymer concentration, the fraction of the drug bonded to the polymer was the highest with PVP. Addition of 20% w/w polymer resulted in ∼65-fold increase in the relaxation time in the PVP dispersion and only ∼5-fold increase in HPMCAS dispersion. In the PAA dispersions, there was no evidence of drug–polymer interactions and the polymer addition did not influence the relaxation time. Thus, the strongest drug–polymer hydrogen bonding interactions in PVP solid dispersions translated to the longest structural relaxation times and the highest resistance to drug crystallization

Influence of Molecular Mobility on the Physical Stability of Amorphous Pharmaceuticals in the Supercooled and Glassy States

We investigated the correlation between molecular mobility and physical stability in three model systems, including griseofulvin, nifedipine, and nifedipine–polyvinyl­pyrrolidone dispersion, and identified the specific mobility mode responsible for instability. The molecular mobility in the glassy as well as the supercooled liquid states of the model systems were comprehensively characterized using dynamic dielectric spectroscopy. Crystallization kinetics was monitored by powder X-ray diffractometry using either a laboratory (in the supercooled state) or a synchrotron (glassy) X-ray source. Structural (α-) relaxation appeared to be the mobility responsible for the observed physical instability at temperatures above <i>T</i>_g. Although the direct measurement of the structural relaxation time below <i>T</i>_g was not experimentally feasible, dielectric measurements in the supercooled state were used to provide an estimate of the α-relaxation times as a function of temperature in glassy pharmaceuticals. Again, there was a strong correlation between the α-relaxation and physical instability (crystallization) in the glassy state but not with any secondary relaxations. These results suggest that structural relaxation is a major contributor to physical instability both above and below <i>T</i>_g in these model systems

Correlation between Molecular Mobility and Physical Stability in Pharmaceutical Glasses

We investigated a possible correlation between molecular mobility and physical stability in glassy celecoxib and indomethacin and identified the specific mobility mode responsible for physical instability (crystallization). In the glassy state, because the structural relaxation times are very long, the measurement was enabled by time domain dielectric spectroscopy. However, the local motions in the glassy state were characterized by frequency domain dielectric spectroscopy. Isothermal crystallization was monitored by powder X-ray diffractometry using either a laboratory source (supercooled state) or synchrotron source (glassy state). Structural (α) relaxation time correlated well with characteristic crystallization time in the supercooled state. On the other hand, a stronger correlation was observed between the Johari–Goldstein (β) relaxation time and physical instability in the glassy state but not with structural relaxation time. These results suggest that Johari–Goldstein relaxation is a potential predictor of physical instability in the glassy state of these model systems

Correlation between Molecular Mobility and Physical Stability in Pharmaceutical Glasses

We investigated a possible correlation between molecular mobility and physical stability in glassy celecoxib and indomethacin and identified the specific mobility mode responsible for physical instability (crystallization). In the glassy state, because the structural relaxation times are very long, the measurement was enabled by time domain dielectric spectroscopy. However, the local motions in the glassy state were characterized by frequency domain dielectric spectroscopy. Isothermal crystallization was monitored by powder X-ray diffractometry using either a laboratory source (supercooled state) or synchrotron source (glassy state). Structural (α) relaxation time correlated well with characteristic crystallization time in the supercooled state. On the other hand, a stronger correlation was observed between the Johari–Goldstein (β) relaxation time and physical instability in the glassy state but not with structural relaxation time. These results suggest that Johari–Goldstein relaxation is a potential predictor of physical instability in the glassy state of these model systems