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Effect of Glass-Forming Matrices on Phospholipid Bilayers during Biopreservation. A 31P NMR Line Shape Simulation Study

Pragati Jain, Sabyasachi Sen, and Subhash Risbud. Chemical Engineering and Materials Science, University of California, Davis, One Shields Ave, Davis, CA 95616

It is well known that certain sugars have the ability to preserve and stabilize membranes, proteins and other living cells in the dry state for extended periods of time. Certain crustaceans, plant seeds and other organisms can even survive dehydration for several years, and resume metabolism upon rehydration. Such organisms accumulate large amounts of disaccharides in their cells, which gives them the capability of undergoing anhydrobiosis, the ability to survive complete dehydration. Other sugars such as lactose, in addition to sucrose and trehalose, are used commercially for freeze-drying biomaterials and products used in the pharmaceutical industry. There are contrasting views on the mechanism that enables disaccharides to stabilize and preserve living membranes. The two principal theoretical models present in the literature to explain this phenomenon are the water replacement model and the vitrification model. Structural and dynamical studies of the atomic-scale interaction of these sugars with the lipid head groups are required to understand the role of these vitreous sugars in the stabilization of biomembranes. 31P nuclear magnetic resonance (NMR) spectroscopy is especially useful in this regard as it can give direct quantitative information about the orientation and dynamics of the lipid head groups. In a previous study we have reported temperature dependent 31P NMR spectra of pure, freeze-dried dipalmitoylphosphatidylcholine (DPPC) lipid membrane and of DPPC in a wide range of glass-forming matrices. Three glass-formers were selected for this study: trehalose is a disaccharide with Tg = 115C, glucose is a monosaccharide with Tg = 30C and hydroxyethyl starch (HES) is a carbohydrate polymer with Tg = 110C. Changes in 31P NMR line shapes as a function of temperature were qualitatively related to the effects of various glass-forming matrices on the mobility of lipid phosphate head groups. However, quantitative studies to determine the effects of glass-forming additives on the orientational and dynamical properties of lipid head groups had not been attempted. Here we report the results of quantitative simulations of temperature dependent 31P NMR line shapes of these systems acquired at temperatures ranging between 25C and 80C. The orientational and dynamical parameters of the DPPC lipid head groups as obtained from these simulations are shown to provide key information about the structural and phase change behavior of the lipid membrane and to lead to a better understanding of the role of sugars in biostabilization. It may be noted here that the selection of these three glass-formers was driven by the significant differences between trehalose and glucose in terms of their Tg and by the contrasting behavior of HES with respect to the sugars in terms of the absence of hydrogen bonding with DPPC in the latter. Such a selection ensures that both the water replacement model and the vitrification model can be adequately tested. Simulations of these line shapes with a model of rotational diffusion of the PO4 headgroups yield dynamical and orientational information regarding the mobility of these head groups as a function of temperature in the presence of two different sugars and one starch. The gel-to-liquid crystalline phase transition of DPPC is characterized by a sudden onset of rapid uniaxial rotational diffusion of the PO4 headgroups. The temperature of this onset of rapid rotational motion shifts to higher values in the presence of glass-forming sugars such as trehalose and glucose. The order of the shift in the transition temperature is glucose > trehalose > HES ≈ pure DPPC while the order of Tg for the glass formers is: trehalose ≈ HES > glucose. Moreover, only trehalose and glucose are found to be able to preserve the initial orientation of the diffusion axes of the lipid headgroups during phase transformation while DPPC/HES behaves in a similar way as pure DPPC. The dynamical and orientational rigidity of the lipid headgroups imparted by the trehalose and glucose matrices are consistent with the presence of hydrogen bonding between the lipid and the sugars and lend support to the water replacement hypothesis of biopreservation.