Theoretical Analysis Of Intracellular Ice Growth And Molecular Dynamics Simulation Of Hydrogen Bonding Characteristics Of Cryoprotective Agent Solutions

Intracellular ice formation(IIF) and ice crystal growth are of great importance in research on cell freezing injury.To understand the exact mechanism of IIF injury,besides IIF and ice crystal growth,the interactions between intracellular ice and cryoprotective agent (CPA),intracellular proteins and cell membrane must be investigated.As a starting work,the structure and dynamics of CPA aqueous solutions must be studied.In the present paper, glycerol is selected as a CPA and mouse oocyte is chosen as a model cell.Karlsson's intracellular ice growth model has been modified and CPA-water binary and CPA-water-sodium chloride ternary solutions have been studied using molecular dynamics simulation methods.A method has been introduced to calculate nucleation temperature of systems with variable compositions.Coupling with the cell dehydration equation,the homogeneous nucleation temperature of intracellular solution has been calculated as a function of time.It has been found that for glycerol-water-sodium chloride ternary solution,the depression of homogeneous nucleation temperature is linearly related with the depression of equilibrium melting point with a factor of 1.17.Then,the relation,coupling with soft impingement effect modification,has been used to modify Karlsson's intracellular ice growth model.Using the modified model,the critical cooling rates and critical CPA concentrations for model cell vitrification have been predicted.The results show that the predicted values of the modified model are in good agreement with the values in literature and are better than the predicted values of Karlsson's model.Further investigation shows that soft impingement effects must be considered when the final intracellular ice volume fraction is larger than 0.1.The hydrogen bonding structure and dynamics in CPA-water binary and CPA-water-sodium chloride ternary solutions have been analyzed.Only O-H...O hydrogen bond exists in binary and ternary solutions.The hydrogen bonding ability of oxygen atom in water molecules(OH2) is larger than that of oxygen atoms in glycerol molecules(O1,O2 and O3).The mean number of hydrogen bonds per OH2 atom decreases as glycerol concentration increases.However,the mean numbers of hydrogen bonds per O1,O2 and O3 atom show no tendency as glycerol concentration increases.The hydrogen bonding abilities of hydrogen atoms in water molecules are slightly smaller than that of hydrogen atoms in glycerol molecules.Unlike oxygen atoms,the difference between hydrogen bonding abilities of hydrogen atoms in water and glycerol molecules are quite small.The mean number of hydrogen bonds per hydrogen atom doesn't change with glycerol concentration.The mean number of hydrogen bonds per glycerol molecule shows a tendency of decrease as glycerol concentration increases,but the tendency is not obvious,considering the errors.The mean number of hydrogen bonds per water molecule decreases as glycerol concentration increases. The results of hydrogen bonding dynamics analysis show that the relaxation time of hydrogen bonding intermittent autocorrelation function and hydrogen bonding lifetime show a tendency of increase as glycerol concentration increases and the lifetime of hydrogen bonds between water molecules is shorter than that of hydrogen bonds between glycerol and water molecules indicating that hydrogen bonds between glycerol and water molecules are stronger than that between water molecules.The hydration structures of sodium cation and chlorine anion are not affected by glycerol molecules relative to other aqueous solutions.The depression of freezing point caused by CPA has been analyzed.The reason is that as CPA is added in,the mean number of hydrogen bonds per water molecule decreases and the lifetime of hydrogen bonds between glycerol and water molecules is larger than that of hydrogen bonds between water molecules.To explore the relation between freezing point depression caused by CPA and the hydrogen bonding network in quantity,a new parameter the ratio between hydrogen bonding acceptor to donor numbers has been introduced.It has been shown that,at low CPA cocentrations,the equilibrium melting temperature T_m is linearly related with the ratio between hydrogen bonding acceptor to donor numbers for water moleculesφ_w.As CPA concentration increases,the linarity betweenφ_w and T_m breaks and T_m becomes linearly related with the ratio between hydrogen bonding acceptor to donor numbers for glycerol moleculesφ_g.The concentration range in whichφ_w(φ_g) is linearly related with T_m depends on the temperature tolerance.When a tolerance of 2K is used,under all hydrogen bonding criteria and at all temperatures,a concentration x_g~* exists:when x_g≤x_g~*, T_m is linearly related withφ_w and when x_g≥x_g~*,T_m is linearly related withφ_g.For binary solutions,x_g~*=0.15 and for ternary solutions,0.13＜x_g~*＜0.15.