Investigation Of The Mechanism Of Cryoprotectant Inhibiting Ice Formation

Cryobiology is an inter-and trans-disciplinary subject involving biology, chemistry, physics and medicine, etc., of which the aim is to study various biological phenomenons and their relationships of living organisms, and to preserve cell, tissue, organ and a whole organism at low temperature for a long term. Cryopreservation is a sub-discipline of cryobiology, which is widely applied to industry, agriculture, medicine, livestock, etc. By means of artificial environment of low temperature, the metabolism of the cells in living organism will be slowed down temporarily, resulting in long-term cryopreservation. Cryoinjury is the biggest impediment to the development of cryopreservation. Thus researchers employ several methods to reduce or eliminate the effect of cryoinjury, of which cryoprotectant is the most effective and widely used one. However, for the application of cryoprotectant, too little cryoprotectant will not take enough cryoprotective effect, and too much cryoprotectant will damage to cells. Thus it is difficult to employ proper amount of cryoprotenctant. This is mainly because that the cryoprotective mechanism of cryoprotectant is not well understood, and it is hard to design a reasonable and effective method of using cryoprotectant. Hence, investigation of the cryoprotective mechanism of cryoprotectant is of significant importance to the improvement of cryopreservation technology, and is an major challenge to the researchers in the field of cryobiology.In the present dissertation, the aqueous solutions of four types of cryoprotectants (including methanol, ethylene glycol, glycerol and dimethyl sulfoxide) are studied by molecular dynamics simulation, and the hydrogen bonding statistics and hydrogen bonding dynamics of the solutions are analyzed. The cryoprotectants help to inhibit the interaction of hydrogen bonding between water molecules, and they also constraint water molecules by hydrogen bonds, resulting in the proportion of "bound water" increases with increasing the concentration. This finding is of significant importance to interpret the the emergence of unforzen water in the freezing process. Moreover, the increasing concentration of cryoprotectant will increase the lifetime of the hydrogen bonds between cryoprotectant and water molecules. This is another indication that cryoprotectant molecule contributes to producing "bound water". Unfrozen water is correlated with the cryoprotectant-water hydrogen bond, which proves that the cryoprotectant-water hydrogen bond is directly responsible for the ice inhibition. This dissertation further analyzes the effects of concentration and temperature on the self-diffusion of water in the aqueous solutions of cryoprotectant. It shows that as the concentration increases and temperature decreases, the self-diffusion coefficient of water molecule has a decreasing tendency. The more hydrogen bonds one water molecule has, the slower its self-diffusion is. Thus the cryoprotenctant molecules slow down the self-diffusion motion of water by hydrogen bonding interaction, resulting in the inhibition of the motion of water towards the ice nucleation. The hydrogen bonded water molecules tend to surround the cryoprotectant molecules, which is benefit to achieving vitrification. This dissertation also analyzes the structures of the hydrophilic and hydrophobic clusters in the aqueous solutions of cryoprotectant, and inteprets the mechanism of the inhibition from the cryoprotectant to water self-diffusion in the point of view of cluter. The hydrophobic cluter is resulted from the attraction between intermolecular alkyl groups, and the cluster size increases with increasing the concentration. It is found that the hydrophobic cluster has significant retardation to the diffusion of water moelcules, and the increasing cluster size with concentration tends to break down the hydrogen bonded water cluster, both of which could effectively reduce the driving force of ice formation.On the basis of the hydrogen bonding analysis of the aqueous solution of dimethyl sulfoxide, the dissertation evaluates the geometric and energetic criteria. Both of the hydrogen bonding criteria are found to have deficiency in the hydrogen bonding statistics, but the effect on the hydrogen bonding analysis could be accepted. Nevertheless, the criteria have large distinction in the analysis of hydrogen bonding dynamics due to that energetic criterion is lack of the limitation to the intermolecular angle of relative orientation. Therefore, extended criterion involving both geometric and engetic thresholds is recommended in the hydrogen bonding analysis of aqueous solution of cryoprotenctant.